Senna auriculata (L.) Roxb. syn. Cassia auriculata L.; Family- Fabaceae ) is a traditional medicinal plant, widely used for the treatment of various ailments in Ayurveda and Siddha system of medicine in India. Almost all the parts of the plant, such as flowers, leaves, seeds, barks, and roots have been reported for their medicinal uses. Traditionally, it has been used in the treatment of diabetes, asthma, rheumatism, dysentery, skin disease, and metabolic disorders. The principle phytochemicals in Senna auriculata (L.) Roxb. are alkaloids, anthraquinone, flavone glycosides, sugar, saponins, phenols, terpenoids, flavonoids, tannins, steroids, palmitic acid, linoleic acid, benzoic acid 2-hydroxyl methyl ester, 1-methyl butyl ester, resorcinol, α-tocopherol-β-D-mannosidase, epicatechin, ferulic acid, quercetin-3-O-rutinoside, quercetin, proanthocyanidin B1. The extracts from its different parts and their isolated compounds possess a wide range of pharmacological activities such as antidiabetic, antioxidant, anti-inflammatory, antihyperlipidemic, hepatoprotective, nephroprotective, cardioprotective, anti-atherosclerotic, anticancer, antimutagenic, antimicrobial, antiulcer, antipyretic, anthelmintic, immunomodulatory, antifertility, anti-venom, and anti-melanogenesis. The toxicological findings from preclinical studies ensured the safety of the plant, but comprehensive clinical studies are required for the safety and efficacy of the plant in humans. The current review article aimed to provide up-to-date information about Senna auriculata (L.) Roxb. covering its ethnomedicinal, phytochemical, pharmacological, and toxicological aspects with special emphasis on its clinical implications in diabetes.Avartaki ( The extensive use of medicinal plants to discover new therapeutics or active pharmacological compounds is the urgent need to tackle challenging non-communicable diseases (NCDs). Around 71% of the mortality worldwide has resulted due to NCDs, including the deaths of 40 million populations worldwide per year . In IndiSenna auriculata (L.) Roxb. syn. Cassia auriculata L., family Fabaceae  is one of the medicinal plants that has been used traditionally in Ayurveda, Siddha, and Unani since the 15th century. It is commonly known as Tanner’s Senna/Cassia and Mature Tea Tree in English; Avartaki, Pitapuspa, Pitkalika, Manojyna, Pitkala, Charmaranga in Sanskrit; Tarwar, Awal, Tarval in Hindi; Tangedu, Merakatangeedu in Telagu, and Arsual, Taravada, Tarwad in Marathi. This shrub is widely spread in India, covering its southern, westerns, and central dry zones and also in Sri Lanka  Roxb., and endeavored to validate the experimental studies in terms of scientific data concerning the therapeutic implications of active metabolites of the plant.The current review aimed to investigate the ethnomedicinal uses, phytochemical, pharmacological, and toxicological studies of the Senna auriculata (L.) Roxb.,” “Cassia auriculata L.,” “Avartaki,” “Avarai,” “Tanner’s cassia,” “phytochemistry,” “antihyperglycemic,” “oxidative stress,” “antidiabetic,” “hepatoprotective,” “insulinogenic,” “anti-inflammatory,” “immunomodulator,” “antioxidant,” “antihyperlipidemic,” “mechanism of action” with their corresponding Medical Subject Headings (MeSH) terms using conjunctions OR/AND. The search was centered on identifying Ayurvedic claims in the available ethnomedicinal, phytochemical, pharmacological, clinical, and safety reports to understand the role of Senna auriculata (L.) Roxb. syn. Cassia auriculata L. in diabetes and other NCDs. The literature search was commenced before April 2021 and searches were limited to the English language.In the present review, the available literature has been explored from Ayurvedic classical texts, various published technical reports, online scientific data by accessing Scopus directory, Google Scholar, PubMed, Science Direct, EMBASE, SciFinder, Web of Science for its ethnomedicinal uses, phytochemistry, pharmacology, and biomedicinal applications. The following keywords were used: “Senna auriculata (L.) Roxb. is found in wooden grasslands up to a height of 500 m. It breeds wild in dry regions with annual precipitation of 300 mm. It grows well in areas with an annual temperature range of 15–28°C and needs full sun for its growth. It is a branched shrub with height of 1.5–5 m, trunk diameter of 20 cm and brown lenticellate bark  and have axillary raceme inflorescence, 2–8 flowered  Roxb. is available in Kaiyadeva Nighantu, a classical Ayurveda text (15th century), where its Pramehahara property (antidiabetic action) through different botanical parts of the plant has been mentioned. Flowers have Pramehashamana property (antidiabetic action). Tender fruits have mentioned for their Vamihara (antiemetic), Krimihara (anthelmintic), Sarvapramehahara (antidiabetic), Trishnaghna , Akshihita (eye tonic), Ruchya (appetizing) properties. Seeds are useful as Madhumehaghna (antidiabetic), Vishahara (antidote), Raktaatisaraghna . Roots are mentioned for Trishnahara , Pramehaghna (antidiabetic), Shwasaghna (antiasthamatic), Raktapittashaman (antihemorrhagic), Shukrakshayahara (sperm enhancing) properties (Senna auriculata (L.) Roxb. was used traditionally to treat inflammation specifically in Maharashtra, Andhra Pradesh, and Gujarat. It also effectively reduces pain and inflammation in joint disorders. The leaves of Senna auriculata (L.) Roxb. are also effective in muscle pain, irregular muscle contraction, body pain, gastritis, skin sores and ulcers. The flowers are effectively used as health beneficial agents. The crushed flowers are mixed with goat milk to cure sexually transmitted diseases. The dried powder of flowers of Senna auriculata (L.) Roxb. is used to clean the hair, and taken by diabetic patients and in fever. The root is used by chewing, and the juice is swallowed to cure abdominal complaints, vomiting, urinary discharges, tumors and diarrhea. Powder of bark is used to treat toothache by applyingit to the gums. The fruits are used in helminthic infections (The foremost description of the operties . The leafections .Senna auriculata (L.) Roxb. reported a significant amount of alkaloids, glycosides, saponins, polyphenols, tannins, phloro-tannins, terpenoids, triterpenes, carbohydrates, proteins, amino acids, anthraquinone, aloe-emodin, and sitosterols. These metabolites attributed towards the pharmacological action in diabetes mellitus and other ailments.The flower, leaves, roots and seed were investigated with advanced spectroscopic and chromatographic techniques and found various active metabolites with therapeutic implications against different diseases. The flowers of Senna auriculata (L.) Roxb. shows the presence of 3-o-methy-d glucose, alpha-tocopherol–beta–D –mannosidase, n-hexadecanoic acid, resorcinol, octadecenal and carboxylic acid. The seeds of Senna auriculata (L.) Roxb. reported with presence of fatty acids content are palmitic, oleic, and linoleic acids. The ethanolic seed extract showed the presence of benzoic acid, 2-hydroxyl methyl ester, glycine, n-(trifluroacetyl), 1-methybutul ester, cupric acid ethyl ester, resorcinol, water-soluble galactomannan like beta-D-manopyranosyl-1(1–4)-o-beta-D-manopyronosyl (1–4)-o-beta–D-monopyranose. The reported metabolites have potential role in oxidative stress, microbial infections and inflammation related diseases.Leaves of Senna auriculata (L.) Roxb. shows the presence of anthraquinone glycosides such as 1,3-dihydroxy-2-methylanthraquinone, 1,8-dihydroxy-6-methoxy-2-methylanthraquinone-3-o-rutinoside, 1,8-dihydroxy-2-methylanthraquinone-3-o-rutinoside, flavone glycoside, and two leucoanthocyanins like leucocyanidin-3-o-rhamnopyroside and leucopeonidin-3-o-1-rhamanopyroside. The list of reported metabolites is enlisted in Roots of Senna auriculata (L.) Roxb. leaves extract has experimentally been proven in different diabetic animal models (Senna auriculata (L.) Roxb. is reported to reduce the blood glucose levels in STZ induced diabetic rats (Senna auriculata (L.) Roxb. leaf showed significant reduction of blood glucose (p < 0.05) at a dose of 150 mg/kg of body weight for 2 weeks in alloxan induced diabetic rats. The results were compared with a standard drug, glibenclamide (Antihyperglycemic activity of l models . The aqutic rats . Furthernclamide .Senna. auriculata (L.) Roxb has various antidiabetic metabolites which are enlisted in Senna auriculata (L.) Roxb.The leaves of Senna auriculata (L.) Roxb. flower has shown promising antihyperglycemic activity in a dose 450 mg/kg body weight which found more significant than the doses of 150 and 300 mg/kg body weight. Similarly, the significant reduction in the urine sugar was found with aqueous extract of Senna auriculata (L.) Roxb. at doses of 150 and 300 mg/kg body weight whereas, at 450 mg/kg dose, sugar was absent in the urine of experimental diabetic rats. The result was more promising than glibenclamide-treated rats (Senna auriculata (L.) Roxb.Aqueous extract of ted rats . Also, JSenna auriculata (L.) Roxb. flower possesses antidiabetic agents such as flavonoids and phenolic acids. The anti-diabetic activity of water-soluble fraction of the ethanol extract was compared with aqueous extract of the flower where it was found that ethanol extract exhibited a more significant reduction in blood glucose level at a dose of 250 and 500 mg/kg of the body weight in alloxan-induced diabetic rats. The highly significant (p < 0.001) results of ethanol extract of the flower might be due to the metabolites present in the water-soluble fraction of the ethanolic extract (p < 0.05) and the results were statistically significant in comparison to the standard glibenclamide (The ethanol extract of  extract . Similarnclamide .Senna auriculata (L.) Roxb. in alloxan-induced diabetic rats to check their anti-hyperglycemic activities. Amongst different extracts, the chloroform extract was reported to have more hypoglycemic effects followed by the water-soluble fraction of ethanolic extract and ethanolic extract. The results found comparable with the standard drug, glibenclamide.Senna auriculata (L.) Roxb. have been isolated and their potential antidiabetic activity has been reported with experimental evidence. Senna auriculata (L.) Roxb. Propanoic acid showed potential hypoglycemic effects both in the bioassay-guided study as well as in vivo study, due to its protein tyrosine phosphatase 1B (PTP 1B) inhibitory action. The isolated phytochemical compound exerted comparable result as that of standard drug, glibenclamide. Senna auriculata (L.) Roxb. Oleanolic acid, a natural triterpenoid present in the aerial part of Senna auriculata (L.) Roxb. (Various active compounds from the flowers of .) Roxb.  exhibite.) Roxb. . FurtherSenna auriculata (L.) Roxb. bark found effective in lowering the blood glucose level. After the administration of the bark extract to diabetic rats, it was observed that the blood glucose level reduced with a deviation of 80.9% when compared that on first day (Senna auriculata (L.) Roxb. bark. The result was compared with hexane, ethyl acetate, and aqueous extracts where methanolic bark extract was found to be more effective in diabetic rats. Senna auriculata (L.) Roxb. Rutin and kaempferol have been reported to exhibit antihyperglycaemic and antioxidant activity in STZ-induced diabetic rats (Senna auriculata (L.) Roxb.The methanolic extract of irst day . Moreovetic rats . Besidestic rats . TherefoSenna auriculata (L.) Roxb. The hypoglycemic role of the ethanolic seed extract in alloxan-induced diabetic rats has been demonstrated at a dose of 400 mg/kg body weight where the result was comparable with that of the standard drug, gliclazide. Also, the urine sugar was found absent in seed extract and gliclazide treated diabetic rats (p < 0.001) blood-glucose-lowering activity in STZ-induced diabetic rats. The result was compared with the standard drug, metformin (250 mg/kg body weight) (Senna auriculata (L.) Roxb. seeds in alloxan-induced diabetic rats. It was found that both the extracts possess significant (p < 0.05) anti-diabetic activity when compared with the standard drug, tolbutamide (250 mg/kg body weight).tic rats . Moreove weight) . Aruna aSenna auriculata (L.) Roxb. is a rich source of antidiabetic metabolites like Linoleic acid, n -hexadecanoic acid, Oleic acid, Epicatechin, Procyanidin B1, Dodecanoic acid, Stearic acid, etc. (Senna auriculata (L.) Roxb. has been reported to reduce blood glucose levels in diabetic patients  Roxb.Although the preclinical data and pharmacological actions of various antidiabetic metabolites support each other, no clinical data is available so far. Further Senna auriculata (L.) Roxb. in a high-fat diet-induced type 2 diabetes mellitus C57BL/6 mouse model. The methanolic extract (150 mg/kg body weight) could reduce the blood glucose level gradually in 8 weeks of the experiment. The results were similar when compared to a metformin-treated group of diabetic mice. The highest amount of total polyphenols was present in the methanolic extract of root than its aqueous, ethanolic, and chloroform extracts. Further, the HPLC profile of polyphenols showed that coumaric acid is present in the methanolic extract of the root, which attributed to the antihyperglycemic activity of Senna auriculata (L.) Roxb. (Senna auriculata (L.) Roxb. may have strong antihyperglycemic effects due to the presence of coumaric acid..) Roxb. . The p-C.) Roxb. . Thus, tSenna auriculata (L.) Roxb. exhibit antidiabetic activities and play a pivotal role in the correction of the pathological mechanism by regulating the metabolic pathways, enzymatic activities, and gene expressions  Roxb. have been reported to possess an insulinogenic action. The different extracts stimulate the insulin secretion by increasing the number of islets and β-cells and it is evidenced with the increased amount of C-peptide and histological pancreatic sections. The administration of the aqueous leaf extract of Senna auriculata (L.) Roxb. demonstrated the insulinogenic action in mildly and severely diabetic rats. The C-peptide level was also found increased. Pancreatic sections have confirmed the increased number of islets and β-cells. The result was similar to that of the standard drug, glibenclamide (Various plant parts of nclamide .Senna auriculata (L.) Roxb. exhibited the antihyperglycemic action by increasing the level of insulin in diabetic rats which resulted into the increased uptake of blood glucose by peripheral tissue (Senna auriculata (L.) Roxb. flower was found significantly effective (p < 0.001) than its aqueous extract (p < 0.05) in reducing the blood glucose due to its insulinogenic action in alloxan-induced diabetic rats (Senna auriculata (L.) Roxb. leaves and flowers extracts administered diabetic rats. Moreover, oleanolic acid, an antidiabetic metabolite present in the aerial part of Senna auriculata (L.) Roxb. may directly improve insulin biosynthesis, secretion, and signaling by modulating the enzymes connected to insulin activities. It also protects the β-cells and preserves their functionality (Senna auriculata (L.) Roxb. flowers.The aqueous flower extract of l tissue . Furthertic rats . Kalaivaionality . SimilarSenna auriculata (L.) Roxb. in STZ-induced diabetic rats. It was found that the number of insulin receptors was increased on erythrocyte receptors membranes in STZ-induced diabetic control rats. The administration of flower extract resulted in increased total erythrocyte receptors membrane insulin binding sites as well as plasma insulin. Senna auriculata (L.) Roxb. flower extract stimulates insulin secretion and increases the number of insulin binding sites.To study insulin interaction in humans, erythrocytes have been used as a cellular model . InsulinSenna auriculata (L.) Roxb. seed extract has also been reported to possess an insulinogenic action in alloxan-induced diabetic rats. The administration of seed extract resulted in increased insulin levels due to the activation of β-cells (S. auriculata. (L.) Roxb. (Senna auriculata (L.) Roxb. bark potentiated the levels of insulin and C-peptide in diabetic rats. The activated remnant β-cells found in histological sections of the pancreas (S. auriculata (L.) Roxb. leaf by its insulin release stimulatory effect in alloxan-induced diabetic rats. β-cells . Dodecan.) Roxb. . Alex etpancreas . Mohan eSenna auriculata (L.) Roxb. demonstrated insulinogenic action. It is due to an increased number of β-cells and regeneration and activation of remnant β-cells. Moreover, the increased level of plasma insulin is evidenced with raised C-peptide level, a part of proinsulin, and a measure of insulin secretion. The metabolites and their pleiotropic mechanisms should also be studied extensively through in vitro and in vivo studies to know the insulinogenic action of Senna auriculata (L.) Roxb.The various extracts of botanical parts of Liver and pancreas ailments hamper the biochemical pathways in the human body and cause deranged glucose metabolism. The liver and pancreas play a vital role in governing carbohydrate metabolism. They maintain blood glucose and also regulate blood glucose supply to other organs.Starch and sucrose are the main components of carbohydrates. The enzyme alpha-amylase of saliva and pancreatic juice decomposes starch into oligosaccharides. The alpha-glucosidase enzyme catalyses the cleavage of glucose from disaccharides and oligosaccharides. Hence, the use of Alpha-glucosidase inhibitors is considered one of the effective treatments of diabetes mellitus as they retard digestion of both sucrose and starch.Senna auriculata (L.) Roxb. have been revealed where the bud extract showed higher inhibition activity compared to flower extract (Senna auriculata (L.) Roxb. showed the concentration-dependent alpha-amylase inhibition activity which found better than standard drug, acarbose  Roxb. bud extract and Senna auriculata (L.) Roxb. flower extract possess glucosidase inhibitory activity. The concentration of the bud and flower extracts shows a direct proportion to the inhibition of α glucosidase enzyme (Senna auriculata (L.) Roxb. was found higher than the standard acarbose (Senna auriculata (L.) Roxb. were found to have a potential alpha-glucosidase inhibitory activity as compared to acarbose in rats. The ED50 of the methanol extracts of dried flowers (4.9 mg/kg) exhibited the antihyperglycemic effect as potent as that of standard drug, acarbose (ED50− 3.1 mg/kg) in maltose loaded Sprague Dawley Rats  Roxb. extracts of acetone, ethanol, and water indicated the presence of flavonoids, tannins, reducing sugar, and saponins. The active components of the extracts compete with the substrate for binding to the active site of the enzyme and prevent the breaking down of oligosaccharides to disaccharides. acarbose .Senna auriculata (L.) Roxb. However, more in vitro and in vivo studies would be helpful to find out the pathways involved in the regulation of enzymatic activities.Altogether, the effective inhibition of alpha-amylase and alpha-glucosidase might be due to the presence of enzyme inhibiting metabolites present in Senna auriculata (L.) Roxb. is reported to play an important role in the activation of hexokinase in alloxan-induced diabetic rats which found resulted in increased glycolysis and glucose consumption for energy production (Senna auriculata (L.) Roxb. stimulates hexokinase activity which aids its role in glycolysis in all body tissues.Enzymatic activity of hexokinase solely depends on insulin response and it is found affected in insulin-deficient diabetic rat liver . Senna aoduction . The insSenna auriculata (L.) Roxb. effectively reduced the gluconeogenesis and resulted in decreased blood glucose level in diabetic rats. The augmented plasma insulin level by Senna auriculata (L.) Roxb. resulted in the reduced levels of glucose-6-phosphatase and fructose-1,6-biphosphatase enzymes in diabetic rats (Senna auriculata (L.) Roxb. bark extract.The role of Glucose-6-phosphatase and fructose-1,6-biphosphatase is also important in glucose homeostasis . These etic rats . Daisy aSenna auriculata (L.) Roxb. has demonstrated the increase in insulin secretion and activation of glycogen synthetase system which resulted in improved liver glycogen content  Roxb. might be responsible for the modifications in enzymatic activities and reducing the blood glucose level (Insulin deficiency in diabetes severely hampers the activities of glycolytic and gluconeogenic enzymes . Insulinse level .Senna auriculata (L.) Roxb. has also been reported for its salutary effect on hepatic enzymes involved in the carbohydrate mechanism. The flower extract (450 mg/kg body weight) was found significant when compared with the standard drug, glibenclamide (600 µg/kg body weight) in increasing the hexokinase activity and decreasing the gluconeogenic enzyme activity in STZ-induced diabetic rats. The increased plasma insulin level in flower extract-fed diabetic rats was responsible for the modulation of hepatic carbohydrate metabolic enzymes The flower extract of Senna auriculata (L.) Roxb. significantly (p < 0.001) elevated the levels of hepatic glycogen and glycogen synthase due to its insulinogenic effects (Senna auriculata (L.) Roxb. bark extract treatment (Senna auriculata (L.) Roxb. reveals its active role in the correction of the deranged carbohydrate metabolism by regulating the various enzymatic activities in the liver (The liver glycogen level and glycogen synthetase system depend on adequate plasma insulin level. The water-soluble fraction of ethanol extract of flower of  effects . Similarreatment . Altogethe liver .Senna auriculata (L.) Roxb. bud extract showed the upregulation of the IRS-2 gene expression in the liver (Senna auriculata (L.) Roxb. flowers were found able to enhance IRS-2, glucose transporters, and Akt gene expression in livers of T2DM rats IRS-2 gene arbitrates insulin activity through the PI3K-Akt pathway in the liver . Also, Ihe liver . Also, pSenna auriculata (L.) Roxb. is used in liver diseases, in the traditional systems of Indian medicine. The folk claims have been validated with different experimental works. The various extracts from the leaves and root of Senna auriculata (L.) Roxb. are reported to exert a hepatoprotective role in oxidative stress-induced, ethanol and anti-tubercular-drug induced, hepatotoxicity in rats (Senna auriculata (L.) Roxb.  Roxb. .Senna auriculata (L.) Roxb. could efficiently (p < 0.001) reduce the AST and ALT levels. Further, the increased values of acid phosphatase (ACP) and alkaline phosphatase (ALP) in alloxan-induced diabetic rats were also declined with the administration of water-soluble fraction of ethanol extract  Roxb. as a hepatoprotective agent. However, there is a need for clinical evidence to confirm and validate the folklore claims, as human studies have not been performed so far.Taken together, these outcomes supports the traditional use of Senna auriculata (L.) Roxb. demonstrated the prophylactic effect on ethanol-induced pancreatitis in a rat model. A significant reduction in the increased pancreatic enzymes such as serum α-amylase and lipase was observed at a dose of 400 mg/kg body weight. Histopathological studies also revealed normal findings in extract-treated rats (The aqueous leaf extract of ted rats .Senna auriculata (L.) Roxb. The isolated compounds such as kaempferol-3-O-rutinoside, quercetin, rutin, and luteolin exhibit different pharmacological actions (Senna auriculata (L.) Roxb., kaempferol-3-O-rutinoside possesses most potential pancreatic lipase inhibitory action (IC50 = 2.9 ± 0.50 mM) than that of rutin, luteolin and quercetin. Moreover, the crude extract of Senna auriculata (L.) Roxb. demonstrated inhibition of pancreatic lipase at IC50 of 6.0 ± 1.0 mg/ml (Senna auriculata (L.) Roxb. (Senna auriculata (L.) Roxb.Several phytochemical compounds have been isolated from different plant parts of  actions . The ant.0 mg/ml . Oleanol.) Roxb. , when ad.) Roxb. . SimilarSenna auriculata (L.) Roxb. leaves extract exhibited hypolipidemic effects by reducing cholesterol, triglycerides and LDL levels and increasing the levels of HDL in diabetic rats. The atherogenic index was also found raised indicating the cardiovascular risk preventive role of Senna auriculata (L.) Roxb. (Senna auriculata (L.) Roxb. has shown its reversal effects on dyslipidemia and apolipoprotein B (The .) Roxb. . In anotrotein B .Senna auriculata (L.) Roxb. demonstrated antihyperlipidemic effects in Triton WR 1339 (300 mg/kg body weight) induced hyperlipidemia in male albino Wistar rats. The flower extract at dose 450 mg/kg body weight/day found more significant (p < 0.001) in reducing the total cholesterol, triglyceride, low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) levels and increasing the levels of HDL than the standard drug, lovastatin. The antihyperlipidemic effects of ethanolic extract of Senna auriculata (L.) Roxb. flowers might be exhibited due to reduced cholesterol biosynthesis by HMG-CoA reductase inhibition in the liver or by up-regulation of LDL receptors in the liver which involve in hepatic cholesterol clearance  Roxb. flowers (300 mg/kg body weight) resulted in the reversal of altered protein and genes expression levels of HMGR, HMGS, SREBP-1c, ACC1, SREBP-2, CYP7A1, and ABCA1 to near-normal levels  Roxb. flower may also capable to oxidize the NADPH, a co-factor in the lipid metabolism. The increased activity of glucose-6-phosphatase reduces NADP+ to NADPH by providing H+ which is helpful in the fat synthesis from carbohydrates. The administration of flower extract reduced glucose-6-phosphatase activity and produced high NADP+ from NADPH which ultimately resulted in down-regulation of lipogenesis  Roxb. flowers in alloxan-induced diabetic rats. It was found that the ethanol extract was exhibited significant (p < 0.001) reduction in the increased levels of triglycerides and total cholesterol than aqueous extract (p < 0.05) in diabetic rats. The restricted cholesterogenesis and reduced fatty acid synthesis were possible involved mechanism with the extracts of Senna auriculata (L.) Roxb. flowers in lowering of total cholesterol and triglycerides level in diabetic rats.Senna auriculata (L.) Roxb. was also demonstrated antilipidemic activity where the significant reduction in cholesterol, triglycerides, LDL and increase in the HDL levels were observed in alloxan-induced diabetic rats (The seed extract of tic rats . Furthertic rats . AlthougSenna auriculata (L.) Roxb. leaves (400 mg/kg body weight) demonstrated potential anti-atherosclerotic action in STZ-induced diabetic rats. The extract suppressed lipid peroxidation and reduced the levels of oxidized LDL, soluble vascular cell adhesion molecule-1 and plasma fibrinogen. The increased serum nitric oxide level was also observed in extract-fed diabetic rats. It was also confirmed in histomorphological studies that the heart myocardium of extract-fed diabetic animals was showing normal morphology, whereas vacuolation was observed in the myocytes of the control diabetic rats  Roxb. leaves may exhibit the significant anti-atherosclerotic and cardioprotective role in diabetic complications. For example, the leaves of Senna auriculata (L.) Roxb. is a rich source of squalene, a naturally occurring triterpenic hydrocarbon  Roxb. However, more research is needed to clarify the effect of extracts of leaves of S. auriculata (L.) Roxb. on the lipid metabolism and dynamics related to atherosclerosis. Overall, the different extracts of Senna auriculata (L.) Roxb. play an important role in the prevention of atherosclerosis due to their anti-hyperlipidemic effect (in vivo studies are available for the antiatherosclerotic effect of Senna auriculata (L.) Roxb. Besides, more preclinical and clinical studies should be conducted to validate its promising protective effect against atherosclerosis associated with metabolic diseases.Lipid peroxidation inhibiting action, free radical scavenging activity, and anti-inflammatory action of different phytochemicals such as flavonoids, saponins, alkaloids and tannins etc. present in the aqueous extract of rocarbon . Liu et c effect . Only a Senna auriculata (L.) Roxb. bark has demonstrated significant antifertility activity in experimental rats at doses of 100 and 200 mg/kg body weight. The oral administration of methanolic bark extract was continued for 7 days during the estrous stage of experimental rats. After dissecting the pregnant rats on day 10, it was observed that the number of corpus lutea reduced significantly, and there was an increased number of resorptions in the extract-fed rats. The effects were dose-dependent, where the extract at a dose of 200 mg/kg showed 100% antifertility activity. Senna auriculata (L.) Roxb. possesses potential antifertility activity, which may exhibit due to its antiestrogenic action (Senna auriculata (L.) Roxb. is reported to have flavoinoids such as luteolin, kaempferol, and quercetin (in vivo. Further studies are required to investigate the role of metabolites present in different extracts of Senna auriculata (L.) Roxb for its antiestrogenic and antifertility activity.In the early stages of pregnancy, the embryo nutrition and prevention of early abortion depend on the proper secretion of estrogen from corpus lutea. The methanolic extract of c action . The extuercetin . Lu et aSenna auriculata (L.) Roxb. plant and been isolated and evaluated for their cytotoxic actions (Senna auriculata (L.) Roxb. leaves has demonstrated cytotoxic effects on human colon cancer cell line HCT 15 and induced apoptosis mediated cell death. The cytotoxicity of the isolated compound was due to its high lipophilicity resulted in the loss of membrane integrity of the cancer cells. The compound was also found to cause membrane disintegration and it was confirmed with the significant release of lactate dehydrogenase (LDH) from damaged cell membrane as a result of its apoptosis (Senna auriculata (L.) Roxb. leaves in human breast adenocarcinoma MCF-7 and human larynx carcinoma Hep-2 cell lines. The extract exhibited a dose-dependent anti-cancer activity with IC50 values of 400 µg in MCF-7 cells and 500 µg in Hep-2 cells. The inhibition of the growth of extract-treated MCF-7 and Hep-2 cell lines was observed due to nuclear fragmentation and condensation followed by apoptosis mediated inhibition of the proliferation of both the cells. In the same way, another isolated compound, 3-O-beta-D-xylopyranosides (triterpine glycosides), form C. dahurica (Senna auriculata (L.) Roxb.  Roxb.  and Actaasiatica , showed asiatica , and MCFasiatica .Senna auriculata (L.) Roxb., due to which it is pretty early to come to any conclusion for its therapeutic implications in cancer patients.However, very few studies were undertaken to establish the cytotoxic effect of Senna auriculata (L.) Roxb. showed significant immunomodulatory effect in aged rats. The administration of extract in aged rats at different doses was found to activate T cell immunity with increased T and B cell percentage accompanied by an enhanced proliferation of splenocytes in both resting and LPS-stimulated cells. The increased number of T cells was further supported by observing the elevated counts of CD4+, CD8+, and CD25+ regulatory cells. Furthermore, the supplementation of polyphenols decreased ROS production by neutrophils in response to phorbol myristate acetate (PMA) and Escherichia coli activation that could conceivably harm multiple biological systems in aged individuals  Roxb. possesses therapeutic activity and shows potential as analgesic, anti-inflammatory, organ-transplantation, and anticancer effects. A report indicated the protective effect of rutin on humoral and cellular immunity in rat model and caused a significant elevation in antibody titer and total antibody levels (Senna auriculata (L.) Roxb. as immunomodulatory agent.Rutin, the major metabolite present in flower extract of y levels .Thus, weSenna auriculata (L.) Roxb. root was evaluated in cisplatin- and gentamicin-induced renal injury in experimental rats. It was observed that the root extract reduced elevated levels of blood urea and serum creatinine effectively at a dosage of 300 and 600 mg/kg body weight in the cisplatin model and a dose of 600 mg/kg body weight in the gentamicin model (The nephroprotective activity of ethanolic extract of in model . The ethSenna auriculata (L.) Roxb. leaves and flowers, at doses ranging from 250 to 600 mg/kg body weight, showed significant antipyretic activity in yeast-induced pyrexia in experimental rats. The effects were comparable to that of a standard drug, aspirin (The fraction of ethanolic extract of  aspirin .Senna auriculata (L.) Roxb. was investigated in different cell lines, such as HeLa, Vero, Crandell Reus feline kidney (CRFK), and HEL cell cultures. The methanolic flower extract showed the strongest antiviral activity against herpes simplex-1 and 2, and moderate activity against vaccinia, vesicular stomatitis, coxsackie, respiratory syncytial, feline corona, feline herpes, parainfluenza, reo-1, sindbis, and puntatoro viruses (Senna auriculata (L.) Roxb. flowers could be a vital source of anti-herpetic compounds possessing antiviral activity against the double-stranded DNA enveloped Herpes simplex virus (HSV -1 and 2).The antiviral activity of methanolic extract of flowers of  viruses . The metSenna auriculata (L.) Roxb. leaves against earthworm was investigated at the dose level of 20, 40, 60 mg/ml. The standard anthelmintic albendazole was used to compare the results. All the extracts showed the concentration-dependent anthelmintic property. Senna auriculata (L.) Roxb. leaves exhibited significant effects (p < 0.05) at the tested concentrations  as determined by the paralysis and death time. Among all extracts, methanolic extract (40 and 60 mg/ml) was reported its efficacy at par in causing paralysis and death of earthworm at all concentrations (Senna auriculata (L.) Roxb. may be attributed to the metabolites especially tannins and phenolic compounds. Tannins act by binding to free protein in the gastrointestinal tract of the host or glycoprotein on the cuticle of the parasite and phenolic compounds by uncoupling oxidative phosphorylation hinder the energy production in helminth parasites (in vivo and in vitro studies are needed to determine the role of metabolites present in different extracts of Senna auriculata (L.) Roxb. and to verify the antihelmintic activity.The anthelmintic activity of methanolic and ethanolic extract of trations . The antarasites . FurtherSenna auriculata (L.) Roxb. in the inhibition of melanogenesis in B16 melanoma 4A5 cells. The presence of auriculataosides A and B (phlegmacin-type anthracenone dimer glycosides) in Senna auriculata (L.) Roxb. indicated in inhibition of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 protein expression. The methanolic extract in the concentration range of 1–100 µg/ml exhibited significant inhibition of melanogenesis. These dimer glycosides were isolated from the methanolic fraction, and their inhibitory activity were detected in the dose range of 0.03–0.3 μM.Senna auriculata (L.) Roxb. as antioxidant agents in several assays such as ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, hydroxyl radical scavenging, phosphomolybdenum reducing power, β-carotene bleaching assay, hydrogen peroxide radical scavenging, metal chelating activity, and deoxyribose degradation (Senna auriculata (L.) Roxb. measured by FRAP and DPPH assay was highest in flower extracts i.e. 161.5 mg AAE/g.The various studies suggested the potency of radation . The antSenna auriculata (L.) Roxb. has potential free radical scavenging action in both 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays (Senna auriculata (L.) Roxb. significantly lowers the TBARS (thiobarbituric acid reactive substances), hydrogen peroxide, and conjugated dienes and exert a potential antioxidant activity. The extracts also increase the important antioxidant enzymes like glutathione, catalase, superoxide dismutase, ascorbic acid, and vitamin E level in rats (Senna auriculata (L.) Roxb. at 400 mg/kg exhibited significant decrease in serum levels of oxidized low-density lipoprotein (Ox LDL) and TBARS (Senna auriculata (L.) Roxb. exhibited potent antioxidant activity when assessed by DPPH radical scavenging, lipid peroxidation, and reducing power analysis (The methanolic and ethanolic extract of ) assays . Jeyasha in rats . The trend TBARS . The alcanalysis .Senna auriculata (L.) Roxb. The highest ferrous ion chelating activity (90.05%) is reported in the extracts of flowers of Senna auriculata (L.) Roxb. (The phosphomolybdenum assay involves the reduction of Mo (VI) to Mo (V) in the presence of antioxidant compound and subsequent formation of a green phosphate Mo (V) complex at acidic pH. The flower extracts (63.8 mg AAE/g) showed higher antioxidant activity of .) Roxb. .Senna auriculata (L.) Roxb. along with above reported studies, supports its medicinal use in diabetes like metabolic diseases. The presence of polyphenols like rutin and kaempferol might be responsible for its free radical scavenging activity. Hence, these scientific evidence propose the possible therapeutic benefits of Senna auriculata (L.) Roxb. in various diseases.Oxidative stress is the main etiology behind many diseases and the antioxidant potential of Senna auriculata (L.) Roxb. in cyclophosphamide induced chromosomal damage in bone marrow cells of albino mice was studied by p < 0.05) against chromosomal aberration due to presence of flavonoids in ethyl acetate extract.The antimutagenic activity of ethyl acetate extract of Senna. auriculata (L.) Roxb. bark on the mutagenicity of Acridine orange (AO) was studied at the concentration of 2.3, 11.4 and 22.8 µM. The probable mechanism of antimutagenic activity of methanolic extract of Senna auriculata (L.) Roxb. bark, could be due to presence of excellent scavengers of reactive oxygen species (ROS) like singlet oxygen and/or superoxide anion radical whichplay a central role in multistage mutagenesis and carcinogenesis (The concentration-dependent inhibitory effect of the methanolic extract of ogenesis .Senna auriculata (L.) Roxb. exhibited the antimicrobial activity against Escherichia coli, Salmonella typhi, Proteus mirabilis, and Klebsiella pneumoniae. The studies on the antibacterial activity of alcoholic and aqueous extracts of flower of Senna auriculata (L.) Roxb. were demonstrated by using Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Salmonella typhi, Salmonella paratyphi A, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Vibrio cholerae, and Shigella dysentrae. The maximum activity was observed against all organisms except Pseudomonas aeruginosa and Klebsiella pneumoniae. The minimum inhibitory concentration ranged between 12.5 and 75 mg/ml depending on micro-organism and various extract. This study confirmed the antimicrobial activity of flower extract of Senna auriculata (L.) Roxb. and reported that it exhibits significant broad-spectrum activity against Bacillus subtilis and Staphylococcus aureus (s aureus .Senna auriculata (L.) Roxb. The chloroform extract of Senna auriculata (L.) Roxb. were shown to possess an antimicrobial activity against 2 g positive and 2 g-negative human pathogenic bacteria, viz. Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and fungus cultures such as Candida albicans and Aspergillus niger. The extract showed antibacterial activity at all concentrations selected, but only the extract with the concentration of 300 µg/ml showed maximum antibacterial activity against all the organisms except Pseudomonas aeruginosa which were comparable with the standard control, amikacin. The antifungal activity of chloroform extract of Senna auriculata (L.) Roxb. revealed significant effect against Candida albicans and Aspergillus niger with the net inhibition zone of 14 and 14 mm, respectively, at 300 µg/ml concentration, which is almost comparable with standard control, ketoconazole used as an antifungal agent  Roxb. was evaluated for antimicrobial activity against P. vesicularis, Streptococcus faecalis, Aeromonas hydrophilia, Salmonella typhae, Staphylococcus cohni, Serratia ficaria, and E. coli at concentration of 12.5, 25, 37.5 and 50 mg/ml. Antimicrobial activity of Senna auriculata (L.) Roxb. was carried out by well diffusion method. The result indicates the saponins rich fraction of roots of Senna auriculata (L.) Roxb. might be exploited as natural drug for the treatment of several infectious diseases caused by these organisms (The saponins rich fraction of roots of rganisms .Senna auriculata (L.) Roxb. by using the agar-well diffusion method. The acetone extract exhibited maximum antimicrobial activity against Pseudomonas aeruginosa, Bacillus pumalis, and Escherichia coli, whereas the ethanol extract showed more antimicrobial activities against Bacillus subtilis and Candida albicans.Further, Senna auriculata (L.) Roxb. has been proven by cited studies but future prospects about the identification of active metabolites for the same is highly suggested, as their mechanism of action will determine its further therapeutic implications.Thus, above data suggested that plant possess a good antimicrobial activity against various strains of pathogenic bacteria and fungus. The ethno-pharmacological utility of Senna auriculata (L.) Roxb. leaves (300 mg/kg body weight) was evaluated against pylorus ligation induced gastric ulcers, and the results were compared with the standard drug famotidine (10 mg/kg body weight) (Senna auriculata (L.) Roxb. leaf extract decreased the number of ulcers in pyloric ligated rats with a significant reduction in the gastric volume, free and total acidity, and ulcerative index.The anti-ulcer activity of methanolic extract of  weight) . It was Senna auriculata (L.) Roxb. leaf extracts viz. methanolic, ethyl acetate aqueous, and hydroalcoholic extracts in inflammation by using carrageenan induced paw edema. The highest inhibitory action in inflammation was exerted by methanolic extract. The methanolic extract showed significant anti-inflammatory potential at 6 h with percentage inhibition of 37% at 250 mg/kg and 31.63% at 500 mg/kg. This effect was attributed due to presence of alkaloids, flavonoids, tannins, and steroids. The ethyl acetate extract of Senna auriculata (L.) Roxb. at 250 & 500 mg/kg showed significant activity in second phase of inflammation induced by carrageenan with percentage inhibition of 34.16 and 30.79% respectively. The aqueous extract of Senna auriculata (L.) Roxb. (250 & 500 mg/kg) showed significant activity at 6 h with percentage inhibition of 31.06 and 30.62% respectively. The hydroalcoholic extract of Senna auriculata (L.) Roxb. in 250 & 500 mg/kg exerted significant activity at 6 h with percentage inhibition of 23.73 and 30.95% respectively. The standard indomethacin showed significant activity maximum at 6 h with percentage inhibition of 42.56%.Senna auriculata (L.) Roxb. has been proven its traditional uses in inflammation and needs further studies involving clinical subjects for its scientific and regulatory approval.The presence of polyphenols in flower viz. rutin is responsible for the suppression of pro-inflammatory mediator’s release and expression of the inflammatory proteins such as adhesion molecules, COX, and NOS . Hence, Senna auriculata (L.) Roxb. and theophylline performed a significant drug interaction. Their concurrent administration resulted in an increased steady-state level (32.5%) of drug theophylline (Senna auriculata (L.) Roxb. tea and antiepileptic drug carbamazepine in experimental rats. The concurrent administration of herbal tea and carbamazepine in rats elevated (47.1%) the blood levels of the carbamazepine significantly than the levels in animals treated with carbamazepine alone. Therefore, the patients who have advised theophylline or carbamazepine should avoid the concurrent use of herbal tea prepared from Senna auriculata (L.) Roxb.flowers to influence the bioavailability of theophylline.The concurrent use of an herbal tea prepared from the flowers of the phylline . SimilarSenna auriculata (L.) Roxb. with metformin. Both these extracts were found safe at tested doses. However, the co-administration of technology-based supercritical extract  and metformin showed a significant decline (60%) in the absorption of metformin. The traditionally prepared hydro-alcoholic extract did not show any change in the pharmacokinetics of metformin. Furthermore, Senna auriculata (L.) Roxb. leaves. The reduced dose of metformin (45 mg/kg) when combined with Senna auriculata (L.) Roxb. leaf extract (500 mg/kg) exhibited a similar blood-glucose-lowering effect of metformin (90 mg/kg) alone.Senna auriculata (L.) Roxb. and various oral hypoglycemic agents to avoid serious adverse events in diabetic patients upon their concurrent use.Further studies are needed to discover the possible pharmacodynamic and pharmacokinetic interactions between Senna auriculata (L.) Roxb. is one of the main ingredients of many Ayurvedic and Siddha formulations available in the market. These formulations are commonly being used in diabetes, hyperlipidemia, obesity, and diabetic-associated co-morbidities. The formulations available in the market are enlisted in .Senna auriculata (L.) Roxb. (leaf) on normal healthy albino Wistar rats at different doses . They showed that the extract did not produce any mortality up to the highest dose tested i.e. 5,000 mg/kg body weight. Also, animals did not exhibit any toxic signs like restlessness, respiratory depression, convulsion or coma.Senna auriculata (L.) Roxb. root suspended in acacia up to a dose 3,000 mg/kg body weight per oral observed that the alcoholic extract of Senna auriculata (L.) Roxb. did not produce any significant changes in the autonomic or behavioral changes including death during the observation period (Senna auriculata (L.) Roxb. The rats did not show any toxic effects as well as any significant variation in their behavior. Acute toxicity studies of ethanol and aqueous extract of Senna auriculata (L.) Roxb. flowers on healthy adult male albino rats at a dose of 100, 500, 1,000, and 3,000 mg/kg body weight indicated the non-toxic nature of extracts in terms of mortality  Roxb. leaves at a dose of 1,000 and 2,000 mg/kg body weight per oral once a daily for 3 weeks. The rats treated with 1,000 and 2,000 mg/kg doses of extract did not show any drug-induced physical signs of toxicity during the complete experimental period and no mortality was noted.Talapotaka churna (4 g TID), a polyherbal formulation containing Senna auriculata (L.) Roxb. as a major ingredient, in Type II Diabetes Mellitus patients. The results were compared with the standard drug, glimepiride (1 mg BD). In the 2 months of the clinical trial, Talapotaka churna improved the symptoms of diabetes such as polyurea, polyphagia, and polydipsia with effective reduction (p < 0.05) in fasting and postprandial blood glucose levels. Further, a significant (p < 0.01) improvement was reported in disturbed biochemical parameters such as serum creatinine, SGOT, SGPT, and lipid profile.Senna auriculata (L.) Roxb. flowers demonstrated a potential anti-hyperglycemic effect in pre-diagnosed type-2 diabetes mellitus subjects. A significant result (p < 0.001) was observed in 30 days of oral administration of flower extract with the effective reduction in fasting as well as postprandial blood glucose level  Roxb. in human participants.Very little clinical data is available, and further extensive clinical studies are needed to support the experimental outcomes of Senna auriculata (L.) Roxb. provides a scientific evidences to explore the clinical implications as antidiabetic, antioxidant, anti-inflammatory, antihyperlipidemic, hepatoprotective, nephroprotective, cardioprotective, anti-atherosclerotic, anticancer, antimutagenic, antimicrobial, antiulcer, antipyretic, anthelmintic, immunomodulatory, antifertility, and anti-melanogenesis potentials with different underlying signaling pathways. The biological potential and mechanisms of action of many metabolites are need to be scientifically investigated for their molecular modes of action and bioactivities. It could provide a lead for further advancement into therapeutics. Well-organized, well-designed, and multicentric clinical studies should be warranted to evaluate the clinical efficacy and safety of the standardized extracts of Senna auriculata (L.) Roxb. carrying pharmacologically active metabolites. There are limited data from clinical reports on diabetes and associated complications. Based on available evidence data, it is advised that Senna auriculata (L.) Roxb. could be used as an adjunct to the current therapy for diabetes and other metabolic disorders.The present review provides comprehensive data, relevant ethnomedicinal uses, details of metabolites, pharmacological activities of crude and various extracts along with pure compounds, marketed formulations, and its safety profile. Investigations on extracts and metabolites of Senna auriculata (L.) Roxb., to date, it's an unexploited plant species in clinical practice. The reason is the scattered experimental data and scarcity of clinical evidence. The present review focuses on addressing the safe pharmacological actions of Senna auriculata (L.) Roxb. on multiple pathways involved in the pathogenesis of diabetes and various other diseases, which also indulges its ethnomedicinal uses in diabetes mellitus. Also, the extensive animal studies involved different extracts of all the botanical parts of Senna auriculata (L.) Roxb., which provides the limelight for the traditional claims and its medicinal uses in various ailments in folkloric practices. The metabolites present in the Senna auriculata (L.) Roxb. provide an ultimate scope for its wide acceptance in the scientific community to discover and produce synthetic molecules as an adjunct to the current therapy in NCDs. Its commercial cultivation and safety pharmacology aspects will contribute to the national economy and would be helpful to reduce the burden of NCDs with its medicinal uses.Though the available experimental evidence insinuates the therapeutic potential of


Mutations in leucine-rich repeat kinase 2 (LRRK2) are recognized as the most frequent cause of Parkinson’s disease (PD). As a multidomain ROCO protein, LRRK2 is characterized by the presence of both a Ras-of-complex (ROC) GTPase domain and a kinase domain connected through the C-terminal of an ROC domain (COR). The bienzymatic ROC–COR–kinase catalytic triad indicated the potential role of GTPase domain in regulating kinase activity. However, as a functional GTPase, the detailed intrinsic regulation of the ROC activation cycle remains poorly understood. Here, combining extensive molecular dynamics simulations and Markov state models, we disclosed the dynamic structural rearrangement of ROC’s homodimer during nucleotide turnover. Our study revealed the coupling between dimerization extent and nucleotide-binding state, indicating a nucleotide-dependent dimerization-based activation scheme adopted by ROC GTPase. Furthermore, inspired by the well-known R1441C/G/H PD-relevant mutations within the ROC domain, we illuminated the potential allosteric molecular mechanism for its pathogenetic effects through enabling faster interconversion between inactive and active states, thus trapping ROC in a prolonged activated state, while the implicated allostery could provide further guidance for identification of regulatory allosteric pockets on the ROC complex. Our investigations illuminated the thermodynamics and kinetics of ROC homodimer during nucleotide-dependent activation for the first time and provided guidance for further exploiting ROC as therapeutic targets for controlling LRRK2 functionality in PD treatment. Gnt1 and Gnt2) with structural motifs contributed from both monomers  with the greatest contribution to kinetic variance  , which cMI and calculated through the marginal Shannon entropy g_correlation tool in Gromacs 3.3 [Therefore, the correlation between pairs of atoms macs 3.3 . The find represents the dimensionality of d = 3.In which 2+ ion) was treated as a set of nodes (assigned to the Cα atom of each residue) connected through edges, which would be drawn between nodes that stayed within a proximity of 4.5 Å for at least 75% of the simulation time. To indicate the relative distance for information transfer through a certain edge between nodes Concepts from network theories ,82 were gncommunities program, which embedded the Girvan-ewman divisive algorithm and used “edge betweenness” that defines the number of shortest pathways across a given edge as an important partition criterion. To obtain the optimal substructure of the network, edges with the highest betweenness would be iteratively removed from the network, and the remaining ones would be recalculated until each node represents an isolated community.The obtained analysis  (CNA) ananalysis ,60,61. TThe Floyd–Warshall algorithm  was used


Antifreeze protein (AFP) is a proteinaceous compound with improved antifreeze ability and binding ability to ice to prevent its growth. As a surface-active material, a small number of AFPs have a tremendous influence on the growth of ice. Therefore, identifying novel AFPs is important to understand protein–ice interactions and create novel ice-binding domains. To date, predicting AFPs is difficult due to their low sequence similarity for the ice-binding domain and the lack of common features among different AFPs. Here, a computational engine was developed to predict the features of AFPs and reveal the most important 39 features for AFP identification, such as antifreeze-like/N-acetylneuraminic acid synthase C-terminal, insect AFP motif, C-type lectin-like, and EGF-like domain. With this newly presented computational method, a group of previously confirmed functional AFP motifs was screened out. This study has identified some potential new AFP motifs and contributes to understanding biological antifreeze mechanisms. Antifreeze protein (AFP) is a proteinaceous compound with improved antifreeze ability in organisms and is widely identified and validated in various species and subtypes ,2. As inIn agriculture, AFPs are applied to maintain crop and fish production in colder climates and as a clinical treatment on hypothermia and cryosurgery against tumors, thereby implying its specific role in biotechnological fields ,7.AFPs are a group of effective proteins with unique antifreeze properties and typical structural and functional characteristics. From the molecular structural aspect, the current AFPs have originated from the sugar-bind domain of C-type lectins (CTLs) that usually depend on calcium ions. Despite the detailed classification of AFPs, their molecular structures still vary; some common structures are shared by almost all AFPs, and some are also found in the original CTLs such as disulfide bridges and cysteine residues . This fiConsidering that AFPs share similar structural properties and are important in the maintenance of biological activity under certain extreme environments, understanding how these materials utilize their typical structural properties to protect living organisms from low temperature and identifying novel AFPs are the two most crucial research directions in this field. Previous studies have partially revealed the biochemical mechanisms underlying AFP protection of living organisms from internal ice formation. AFPs help regulate the freezing point of certain aqueous solutions to provide a soluble and liquid microenvironment for essential biochemical reactions as firstly reported and confirmed in Dendroides canadensis . In 2014Depending on their specific molecular structures, AFPs have varying participation in regulating the antifreeze properties of organisms. One of the largest obstacles in current AFP studies is the prediction of novel potential AFPs. Although AFPs share some simple structures such as disulfide bridges and cysteine residues, their sequence similarity is low. Hence, predicting novel AFPs according to previous computational methods is difficult. In this study, a computational engine is developed to predict AFP features. The 39 most important features for AFP identification have been revealed. Candidate features and signatures are extracted from a website called “InterPro protein sequence analysis & classification”. These entries are converted to their protein domain by relying on features that represent protein families, domains, repeats, and specific functional sites. Based on the 39 features, the most common protein domains are antifreeze-like/N-acetylneuraminic acid synthase C-terminal, insect AFP motif, and CTL-like domain. This finding indicates that many important key features originated from the protein domains such as insect AFP motif and CTL-like domain. In addition, a group of previously confirmed functional AFP motifs is screened out by using the newly presented computational method. This study has identified some potential new AFP motifs and contributes to the understanding of biological antifreeze mechanisms.A total of 9083 non-AFP and 464 AFP sequences were downloaded from Yang et al.  and are x and y, their MI value can be computed byp is their joint probabilistic density, and p(x) and p(y) are their marginal probabilistic densities. The mRMR method outputs a feature list, named mRMR feature list, to indicate the importance of features. At first, this list is empty. Features are selected one by one and appended to this list. For each of the non-selected features, calculate its relevance to class labels, measured by MI of it and class labels, and its redundancies to already-selected features, evaluated by the average MI values of it and each already-selected feature. The difference between the relevance and redundancies is further computed and a feature with a maximum difference is picked up. This procedure stops until all features have been selected. For formulation, the feature list is denoted asN is the number of all input features. Features that have high ranks are more important than those with low ranks.Lots of domains were used to encode one AFP or non-AFP. Not all domains were highly related to the determination of AFPs. To extract highly related domains, thereby building an efficient classifier, a feature selection procedure was necessary. Here, we selected the widely used minimum redundancy maximum relevance (mRMR) ,14,15,16F, a series of feature subsets The mRMR method produced an mRMR feature list. According to the idea of mRMR, some top features can comprise the optimum feature subsets for a given classification algorithm. Thus, the IFS method  was empllidation ,24,25,26N samples was repeatedly sampled for B times (B is the parameter representing the number of decision trees). For each decision tree, the randomly selected N samples with replacement constituted its training set, and a random feature subset was adopted to split the nodes of this decision tree. B decision trees were eventually grown. For a new sample, each decision tree provided a predicted class, and the predicted class of the RF was determined by majority voting. RF has been applied to tackle many biological problems [As mentioned in problems ,34,35,36The classifiers named “RandomForest” in Weka  with defAs mentioned in In this study, we used functional domains to represent each protein and proposed an RF classifier to identify AFPs. Furthermore, some essential domains were extracted. The entire procedures are illustrated in 13729 domains were used to represent AFPs and non-AFPs. We first used the mRMR method to evaluate the importance of all features, obtaining an mRMR feature list. Such a list is provided in X-axis and MCC was set as Y-axis. It can be observed that such a curve first followed an increasing trend and after it achieved the highest point, it followed a slightly decreasing trend. The highest MCC was 0.937, which was obtained by using the top 39 features. Accordingly, these 39 features were termed optimum features and the RF classifier with them was called the optimum RF classifier. The other three measurements  of such a classifier were 0.890, 1.000, and 0.995, respectively. These results indicated the good performance of such an RF classifier.The mRMR feature list was fed into the IFS method. Because lots of domain features were involved, the IFS method would be time-consuming if all possible feature subsets were considered. To save time, we only constructed the top 500 feature subsets and built an RF classifier on each subset. All classifiers were evaluated by 10-fold cross-validation. Predicted results were counted as four measurements computed by Equations (3)–(6), which are available in The performance of the proposed RF classifier was compared with those of other state-of-the-art methods to reveal its advantages. Given that AFP-Ensemble  does notWith the above arguments, the proposed classifier was superior to previous methods. The most previous methods directly extracted features from protein sequences, which contained limited function information of proteins. The functional domain is much more powerful to identify different properties of proteins because it directly contains the function information of proteins. Although AFP-Ensemble  also empIn the proposed RF classifier, each protein was represented by 39 features. These features were derived from 39 functional domains. According to the construction of such a classifier, these domains were highly related to AFPs. They were classified into seven subgroups for analysis: (1) CTL domain, (2) sushi/SCR/CCP domain, (3) antifreeze-like/N-acetylneuraminic acid synthase C-terminal domain, (4) insect AFP, (5) type I AFPs, (6) type II AFPs, and (7) type III AFPs. The detailed structure information link from the Interpro database and the cluster of each identified domain are provided in CTL domain was matched by eleven InterPro entries and therefore is an important feature for AFP detection. CTLs are generally made up of 110–130 residues. The four typical cysteins are conserved at the structural level and involve a functional two-disulfide bond. Such conserved domains are shared in multiple protein subtypes. As a general cluster of AFPs, type II AFPs have been confirmed to be homologous to such calcium-dependent CTLs in a widely cultivated fish named Osmerus mordax . TherefoAFPs were assumed to have derived from lectin-associated residues in vivo . The con2+ concentration to affect the IRI activity without inducing sequence homology to AFPs. Hence, additional non-AFPs may exist with the ability to display calcium-dependent ice recrystallization inhibition.Some typical peptide sequences and spatial structures do participate in the inhibition of cellular freezing and thus may partially reveal potential AFP mechanisms . For insAmong these domains, three of them are just c-type lectin-like domains , which are CTL domains. Apart from that, some additional domains like link domain (IPR000538), EGF associated domains , and a lectin associated domain have also been clustered into CTL domains. Considering the anti-freeze capacity of CTL domains and EGF domains have been validated in Sushi/SCR/CCP domain was found by two InterPro entries in numerous complement and adhesion proteins. This domain has typical structural properties that reflect its effective biological functions. For each CCP domain, approximately 60 residues have formed a similar domain with CTL ,43. ConsAntifreeze-like/N-acetylneuraminic acid synthase C-terminal domain, also called antifreeze-like protein (AFLP), is an important feature to differentiate AFPs from other proteins. AFLPs are similar to AFPs. Early in 2006, the structure of AFLP was identified and validated by one of the most advanced techniques, nuclear magnetic resonance spectroscopy. Composed of 75 amino acid residuals, the AFL domain is the core functional residuals for AFLPs . SimilarInsect AFP was matched by five InterPro entries and identified in insects. The core regulatory mechanism for insect AFP is the regulation of freezing points on aqueous solutions . For insType I, II, and III AFPs are the most common subtypes. Type I AFPs are a group of gas hydrate crystal inhibitors, and their antifreeze ability relies on their binding capacity against the ice plane. Type I AFPs contribute to the regulation of intracellular antifreeze biological processes as confirmed in multiple artificial and natural environments ,48, indiCompared with type I AFPs, type II AFPs have different natural distribution patterns and are found in species such as sea ravens, smelt, and herring. With regard to structure, type II AFPs can also be regarded as the evolved CTLs with calcium ion-dependent functions. In a natural environment, the antifreeze capacity of herring and smelt can be attributed to type II AFPs , indicatDifferent from type II AFPs that have evolved from CTLs, type III AFPs are derived from a sialic acid synthase gene according to an evolutionary genomic study on Antarctic eelpout . A groupPrevious works have confirmed these predicted high-ranking sequence features (motifs) of AFPs, thus conforming to our prediction. Therefore, the present study has developed a novel computational approach for the detection of potential AFPs and helps us partially reveal the common structural characteristics of anti-freeze associated proteins.This study used functional domains to represent each AFP or non-AFP. Then, some machine learning methods were applied to identify AFP-associated protein domains. According to recent publications, several identified domains with reliable annotation are associated with seven clusters of AFPs. These results can help us take the initial step to understand the biological mechanisms of AFPs at the structural level. Moreover, the classifier using identified domains provided better performance than some previous methods, which can be a useful tool for identifying latent AFPs.


Phyllanthus niruri (P. niruri) or Dukung Anak is a herbal plant in the Phyllanthaceae family that has been used traditionally to treat various ailments such as diabetes, jaundice, flu and cough. P. niruri contains numerous medicinal benefits such as anti-tumor and anti-carcinogenic properties and a remedy for hepatitis B viral infection. Due to its beneficial properties, P. niruri is overharvested and wild plants become scarce. This study was conducted to develop an appropriate in vitro culture protocol for the mass production of P. niruri. An aseptic culture of P. niruri was established followed by multiplication of explants using different types of basal medium and its strength and plant growth regulators manipulation. This study also established the induction of in vitro rooting utilizing various types and concentrations of auxin. Treatment of Clorox® with 30% concentration showed the lowest percentage (%) of contamination, 4.44% in P. niruri culture. Nodal segments of P. niruri were successfully induced in full-strength of Murashige and Skoog (MS) basal media with 2.33 number of shoots, 3.11 cm length of shoot and 27.91 number of leaves. In addition, explants in full-strength MS media without any additional cytokinin were recorded as the optimum results for all parameters including the number of shoots (5.0 shoots), the length of shoots (3.68 cm) and the number of leaves (27.33 leaves). Treatment of 2.5 µM indole-3-butyric acid (IBA) showed the highest number of roots (17.92 roots) and root length (1.29 cm). Rooted explants were transferred for acclimatization, and the plantlet showed over 80% of survival rate. In conclusion, plantlets of P. niruri were successfully induced and multiplied via in vitro culture, which could be a step closer to its commercialization. Phyllanthus niruri (P. niruri), identified locally as Dukung Anak [Over the past several years, medicinal plants have gained greater attention and recognition in Malaysia, particularly as demand for alternative medicine and natural health products increases in 2019 . In addiung Anak .P. niruri (Phyllanthaceae) has traditionally been used to treat several ailments such as jaundice, kidney stone, flu, fever, and diabetes [P. niruri possesses anti-tumor, anti-oxidant, anti-carcinogenic, and hepatoprotective properties [P. niruri that can inhibit the binding of human immunodeficiency virus Rev protein (HIV REV) [diabetes ,6,7. Sevoperties ,9,10. Inoperties . FurtherHIV REV) . Hence, The main issue for the commercialization of herbal-based products is the uniformity and consistency of the planting materials . In addiP. niruri shoots (15.28 ± 0.96) on MS medium supplemented with BAP (0.5 mg/L). In addition, a maximum number of P. niruri shoots (3.16 ± 0.16) was achieved from nodal explants inoculated on MS medium supplemented with BAP (3.0 mg/L) [Plant micropropagation may serve as an alternative solution to ensure the sustainable supply of plant materials. It is an excellent technique to produce plants in a large amount in a short time . Micropr.0 mg/L) .Appropriate selection of chemical sterilization is essential in the first stage of plant micropropagation. All microorganisms that potentially could contaminate the culture should be removed and eliminated . This stCulture medium supplemented with plant growth regulators; cytokinin and auxin were used in many plants to propagate via in vitro techniques ,29. AuxiP. niruri has captured people’s attention worldwide. The plant was harvested with no sanitary consideration taken on the collected samples. The explant obtained from wild or greenhouse is typically contaminated with microorganisms such as bacteria and fungi [Due to its high medicinal values and properties, nd fungi . In vitrnd fungi . The micnd fungi .P. niruri in Malaysia, suggesting that the plant has not been thoroughly utilized for commercialization, especially in Malaysia. Thus, this study may propagate P. niruri rapidly and serve as a preliminary analysis for future studies. The objectives of this study were to examine the effect of different types and concentrations of chemical sterilants on surface sterilization and examine the growth responses of P. niruri explants to different basal media and their strength and plant growth regulators.Besides, there is no information on the production of P. niruri were cultured on MS medium without any plant growth regulators following the surface sterilization procedure. There were significant differences between different chemical sterilants and their concentrations on the percentage of contamination of explant of P. niruri. Different concentrations of nanosilver showed no significant effect on their percentage of contamination  and Gamborg B5 (B5) basal media markedly influent the growth responses of P. niruri explant. This can be seen in Similarly, explants in half-strength B5 basal media generated the number of shoots, length of shoot, and the number of leaves  that were not significantly different from those explants developed in full strength B5 basal media treatment .In contrast, all parameters in higher strength of MS basal media (double strength) recorded a significantly lower number of shoots (0.67), shorter length of the shoots (1.5 cm), and fewer number of leaves (4.88) compared to the full strength of MS basal media. The increasing strength of B5 basal media, from full- to double-strength, also reduced the number of shoots with their respective values of 3.00 and 1.67. Likewise, the length of shoot and number of leaves also lessen significantly when the media strength was amplified to double strength .The nodal segment of P. niruri showed a great response in media supplemented with cytokinin along with control treatment. The inoculated P. niruri in MS media with cytokinin treatment showed signs of axillary buds’ proliferation a as earlResults in The media containing auxins induced a higher number of roots than the control treatment. In vitro regenerated plantlets adapted well to acclimatization as plantlet leaves grow more prominent and thicken with new leaves emergence from day 8–15 b. Pottin®, a conventional bleach that consists of 5.25% of sodium hypochlorite (NaOCl), has the ability to penetrate the inner layers of the plant. It has been extensively used along with ethanol as an effective surface sterilizing agent in various plant species such as Ludisia discolour [Gerbera hybrida [Ananas comosus [Jatropha curcas [Aquilaria malaccensis [Taraxacum belorussicum [Preventing fungal and bacterial contamination is very critical to ensure the success of micropropagation. The least percentage of contamination of explants indicated the chemical sterilants’ efficiency to remove the microorganism and establish a “clean” culture. Cloroxiscolour , Gerbera hybrida , Ananas  comosus , Jatropha curcas , Aquilaraccensis , sour chaccensis  and Tararussicum .® was chosen as the best surface sterilization treatment since it had the least contamination percentage. Admitting a higher concentration of bleach might diminish the contaminants, but it might induce tissue damage in the meantime [Phyllanthus caroliniensis was established in a study conducted by Catapan et al. [Treatment of 30% Cloroxmeantime . The asen et al.  using 2.However, working with another variety, Ana et al.  indicateOcimum basilicum L. seed yield [3) caused 100% oxidation of Colobanthus quitensis explants. In addition, a higher concentration of AgNO3 did not prevent the appearance of contaminants while a lower concentration did not promote tissue proliferation [3 is varied in its shapes and sizes [Nano silver was found to be an ineffective sterilant in this study. This was expected as all concentrations of nano silver gave no significant difference in the result. However, the findings differed with Rostami and Shahsavar , who suged yield . The stued yield  however feration . This isnd sizes . Thus, tnd sizes .P. niruri explant as it displayed the best result in all parameters. Meanwhile, double-strength medium, regardless of the type of basal medium; MS or B5, produced the lowest results in all growth response parameters. This finding indicated that the increment of nutrient concentration inhibits the growth responses of P. niruri. This study’s results were in agreement with Rezali et al. [Typhonium flagelliforme. These present results are equivalent to the findings reported by Catapan et al. [Phyllanthus urinaria. MS medium was also shown to be the most suitable basal medium for the development of in vitro tubers of Gloriosa superba, while the B5 medium was not at all effective in induction of secondary tubers in vitro [Consequently, the full strength of MS medium (control) in this current study was found to be the most effective basal medium for the development of i et al. , who stan et al. , who proin vitro . This diin vitro , who pro3−) and ammonium (NH4+). MS medium contains two nitrogen salts, ammonium nitrate (~1650 mg/L) and potassium nitrate (~1990 mg/L), for a total nitrogen content of ~3550 mg/L [Phyllanthus amarus [Various responses from explant in producing in vitro explant on the above-mentioned basal media can be due to changes in the basal salt formulation. Nitrogen is an important component in the mineral salt formulation and it can be found in the forms of nitrate . A similar pattern was observed in MS double strength treatment where there was a decrement in the number of shoots and length of shoot of P. niruri. This proven that the double-strength of MS basal media inhibited the growth and development of in vitro P. niruri.The number of leaves is another important parameter to consider. This is because, for certain plant species, higher shoot numbers and length of shoot do not correspond to higher leaves numbers . Based oP. niruri explants can regenerate and produce shoots rapidly on their own, without any additional cytokinin. Presumably, in tissues that can grow without cytokinin being added (exogenous) to the medium, the cells can produce sufficient natural (endogenous) cytokinin for cell division to proceed.This showed that the P. niruri cultured on MS medium fortified with 1.0 mg/L BAP produced the most number of shoots (6.6 shoots) and a combination of Kn and BAP induced multiple shoot formation in all nodal segments. Rajasubramaniam and Saradhi [Phyllanthus fraternus. The application of cytokinin is essential in single node culture, according to Gallavotti [This result differed with the finding by Liang and Keng , who sta Saradhi  also prollavotti , as it hThe endogenous amount of cytokinin present within the explant may be sufficient to induce positive organogenesis. The present study’s findings are in line with Zazimalova et al. , who hav2) and ethylene (C2H4) [The difference between the results might be due to different levels of endogenous PGRs within the explants and exogenous factors such as light intensity, temperature, and relative humidity within the culture room . The groe (C2H4) . In addie (C2H4) ,74.P. niruri explant, without the intermediate callus phase. This was possible because of the totipotent and plasticity nature of P. niruri explant, which initiates the cell division hence regenerates the primary shoot.In general, the organogenic ability of an explant is highly dependent on totipotency and plasticity. In this research, one significant finding was that the primary shoots in medium supplemented with exogenous cytokinin were directly induced axillary buds from the nodal segment of P. niruri was successfully achieved after four weeks of culture.Root induction and growth at the base of in vitro grown shoot are an essential and vital stage in plant micropropagation establishment of plantlet before transferring to the field. For this purpose, the application of auxin as a vital hormone for rooting initiation is necessary. Root production of Phyllanthus urinaria as compared to IAA and NAA treatments. In contrast, working with the same variety, Kalidass and Mohan [Of the auxins tested, 2.5 µM of IBA was the best for proper rooting, in which it produced the highest number of roots compared to other treatments. Tanimoto  has shownd Mohan  recordednd Mohan .Olea europaea L. cv. “Moraiolo” by Ansar et al. [The highest length of the root was found in media supplemented with 2.5 µM of IBA with 1.29 cm while retaining no significant difference with IBA 1.25 µM (1.07 cm) and NAA 2.5 µM (1.22 cm). Another study was performed on r et al.  producedr et al.  proved tr et al.  revealedr et al.  reportedAcclimatization is an essential step in plant micropropagation because it ensures that plantlets grown in vitro adapt to their natural environment when transplanted into nature. These plantlets must adjust to environmental conditions, including humidity, temperature, photoperiod, and pH gradually. As per Kirdmanee et al. , the numThe current study found that the mixture of coco peat and peat moss enhanced the plantlets’ quality, allowing them to withstand the harsh external climate. The results were in accordance with that of Manjusha and Sathyanarayana , who alsP. niruri, which produced the highest percentage of plantlet survival.After 4 weeks under ex vitro conditions, the plantlet survival rate was over 80%, demonstrating that this procedure and protocol can be utilized as an efficient method for plant acclimatization. Furthermore, the ex vitro established plantlets displayed normal growth characteristics with no noticeable abnormalities. Hence, potting media treatment of coco peat + peat moss was optimal for acclimatization of P. niruri plant was collected from the UPM compound and garden. A voucher specimen of P. niruri was deposited at the Institute of Bioscience, UPM, to identify and confirm plant species and given the voucher number SK 3356/18.The explants were obtained by excising the stem nodes of P. niruri. The stem nodes were cut into 1–1.5 cm long and washed with detergent for 30 min under running tap water. Thereafter, the surface sterilization procedure was conducted under a sterilized condition in a laminar flow hood. The explants were sterilized using 70% ethanol for 30 s then washed with sterile distilled water at least three times. From here on, the explants were submerged into different chemical sterilants  with an addition of 2–3 drops of Tween 20 surfactant and shaken for 30 min. After that, all the treatment solutions were discarded, and the explants were rinsed at least three times with sterile distilled water.The parameter taken for this experiment was the percentage of contamination by counting the number of contaminated explants in each flask at the end of the second week after cultivation. The flask containing cultured explants was incubated for two weeks at a temperature of 25 ± 3 °C under 16 h light and 8 h dark of photoperiod alongside white fluorescent light radiation of 45 µmol/m²/s in the incubation room.From the counting, the percentage of contamination was measured according to the formula:Two types of basal media prepared using media formulation were explained by Murashige and Skoog  containing iron chelated to the disodium salt of EDTA and Gamborg et al.  which containing iron chelated to the mono-sodium salt of EDTA. Briefly, the macronutrients, micronutrients, and vitamins solution was mixed with 0.1 g/L of myo-inositol  and 30 g/L of sucrose . The medium’s pH was adjusted to pH 5.6–5.8 by adding 1 M of either sodium hydroxide (NaOH) and hydrochloric acid (HCl). A total of 3 g/L of Gelrite™  was added and stirred until completely dissolved. The solution was heated in the microwave before being poured into vials. All the labeled vials were then placed in the autoclave to be sterilized at 121 °C and 1.05 kg/cm² for 20 min The strength of basal media  was manipulated by either reducing or amplifying the macronutrients, micronutrients, and vitamins. MS with full strength served as a control treatment in this experiment. The data were taken on the number of shoots, length of the shoot (cm), and the number of leaves was recorded after four weeks of incubation.The best basal medium and its strength from the previous experiment were prepared and supplemented with different cytokinin; 6-benzylaminopurine (BAP), kinetin (Kn), Zeatin (Zn), and 2-isopentenyl adenine (2iP)  at different concentrations of 2.5, 5, 7.5, 10.0 µM. The control treatment was devoid of any plant growth regulators. The cultures were maintained for four weeks of incubation. The parameters taken for this experiment were the number of shoots, length of the shoot (cm) and the number of leaves. All the data collection was carried out after four weeks of culturing.The explants from the previous experiment were subcultured to a fresh MS media containing different types of auxin at different concentrations. The basal media was supplemented with different auxin; indole-3-butyric acid (IBA), 1-naphthalene acetic acid (NAA), and indole-3-acetic acid (IAA) at different concentrations of 1.25, 2.5, and 5 µM. Media without any auxin serve as control treatment. The parameters taken for this experiment were the number of roots and length of root (cm). All the data collection was carried out after four weeks of culturing. Root induction and development parameters were taken on week four of inoculation.Rooted explants were removed and rinsed gently with distilled water to remove any agar trace before transferring to pot under ex vitro condition in potting media containing peat moss (Hup Nong Agriculture Sdn. Bhd.) and cocopeat (Cofibers Sdn. Bhd.) at ration 1:1. Any trace of agar might encourage fungal and bacterial infections. The plantlets were kept in a shaded area and watered daily for four weeks. The plastic cover was gradually removed after the first week, and plantlet were exposed to 70% humidity and temperature of 28 ± 4 °C.The percentage of survival was calculated using the formula as below:p < 0.05.All experiments were conducted in a completely randomized design and were repeated twice. Each treatment consisted of three replicates. Mean values of various treatments were subjected to analysis of variance (ANOVA), and the significant difference was separated using Least Significant Difference (LSD). SAS version 9.4 was used to determine the significance at P. niruri. The establishment of aseptic culture was achieved using 30% of Clorox® as a sterilizing agent to diminish microbial contaminations. Various plant growth factors which including basal medium and plant growth regulators were studied to find the best formulation for the growth and development of P. niruri. According to the findings, full-strength MS basal medium with no cytokinin content (control) treatment produced the best results with a high number of shoots, length of shoot and number of leaves. The root induction of P. niruri explant has been influenced by different types and concentrations of auxins, as the result showed the media supplemented with 2.5 µM of IBA exhibited the best result, which produced 17.92 roots and 1.29 cm length of roots after four weeks of incubation.This current study suggested an efficient protocol for in vitro propagation of P. niruri plantlet in a media mixture of coco peat and peat moss at a ratio of 1:1. This study has effectively developed a protocol for propagating P. niruri through plant tissue culture. This research can therefore contribute to the production of P. niruri raw materials to meet the requirements of the herbal industry. Originally, it was assumed that all plants regenerated from cell or tissue culture would have genetic materials identical to the parent plant. Despite this, phenotypic variation must be common between regenerated plants. Thus, further studies on DNAs of regenerated plantlets can be conducted to determine its somaclonal variation level when compared to the mother plant.Besides that, ex vitro establishment was achieved with an 88% of survival rate of


SorLA is a member of the Vps10p-domain (Vps10p-D) receptor family of type-I transmembrane proteins conveying neuronal endosomal sorting. The extracellular/luminal moiety of SorLA has a unique mosaic domain composition and interacts with a large number of different and partially unrelated ligands, including the amyloid precursor protein as well as amyloid-β. Several studies support a strong association of SorLA with sporadic and familial forms of Alzheimer’s disease (AD). Although SorLA seems to be an important factor in AD, the large number of different ligands suggests a role as a neuronal multifunctional receptor with additional intracellular sorting capacities. Therefore, understanding the determinants of SorLA’s subcellular targeting might be pertinent for understanding neuronal endosomal sorting mechanisms in general. A number of cytosolic adaptor proteins have already been demonstrated to determine intracellular trafficking of SorLA. Most of these adaptors and several ligands of the extracellular/luminal moiety are shared with the Vps10p-D receptor Sortilin. Although SorLA and Sortilin show both a predominant intracellular and endosomal localization, they are targeted to different endosomal compartments. Thus, independent adaptor proteins may convey their differential endosomal targeting. Here, we hypothesized that Sortilin and SorLA interact with the cytosolic adaptors PSD95 and PICK1 which have been shown to bind the Vps10p-D receptor SorCS3. We observed only an interaction for SorLA and PICK1 in mammalian-two-hybrid, pull-down and cellular recruitment experiments. We demonstrate by mutational analysis that the C-terminal minimal PDZ domain binding motif VIA of SorLA mediates the interaction. Moreover, we show co-localization of SorLA and PICK1 at vesicular structures in primary neurons. Although the physiological role of the interaction between PICK1 and SorLA remains unsolved, our study suggests that PICK1 partakes in regulating SorLA’s intracellular itinerary. SorLA  a transmembrane protein with a short cytosolic and a large extracellular/luminal part is prominently, but not exclusively, expressed in the brain . It consSORL1 with both sporadic and familial forms of Alzheimer’s disease (AD) [Genetic analysis revealed an association of ase (AD) . AD braiase (AD) , which wase (AD) , 19. In ase (AD) . In neurase (AD) , 20, 21.ase (AD) , 22. TheSorLA locates to the TGN and endosomes and only a minority is found on the cell surface , 23, 24.Cellular uptake of ligands conveyed by SorLA is rather slow when compared to LDLRs such as LRP1 . The cytTo date, only a few adaptor proteins have been described to bind the cytosolic domains of the SorCS subset of Vps10p-D receptors. SorCS1 interacts with AP-2 that likely mediates the internalization of all Vps10p-D receptors . The SorPICK1 has a unique domain composition with a single N-terminal PDZ domain followed by a BAR (Bin-Amphiphysin-Rvs) domain , 36. PICThe PICK1 PDZ domain tethers transmembrane proteins as cargo and the BAR domain binds curved-membranes which can bud and form transport vesicles. It becomes fully functional through homo- or hetero-dimerization with another BAR domain protein. PICK1 constitutes a heteromeric BAR-domain complex with ICA69 , 48. TheAlthough the physiological significance of the interaction of PICK1 and several of its ligands remains poorly understood, PICK1 regulates the trafficking of transmembrane proteins through binding their C-termini with its PDZ domain.53 or V52 and I53 to alanine  to alanine (T70mV-SorLA-CD-VAA) strongly reduced, but did not impede completely the recruitment of tdTomato-PICK1  to alanine (IL2R-SorLA-CD-VAA) resulted in a similar subcellular localization as compared to IL2R-SorLA-CD  does not change its internalization rate in CHO cells [A previous study showed that the truncation of the terminal four amino acids of SorLA -X-Φ, II interacts with Φ-X-Φ, III interacts with Ψ-X-Φ, and IV which interacts with (D/E)-X-Φ as preferred C-terminal motif, where X is any residue, Φ is a hydrophobic residue and Ψ is a basic hydrophilic residue . Most PD12ANSHY17 motif with retromer [30–D38 facilitates binding of AP-1, AP-2 and PACS1, the heat shock protein HSPA12A binds the SorLA-CD via the acidic residues E34-D38 and D47D48 in an ADP/ATP-dependent manner and D47DXXM is regarded as the minimal GGA interaction motif  is crucial for SorLA’s internalization, TGN to endosome transport and polarized neuronal targeting [48XXM51 [52-A54). Truncation of the terminal four amino acids (M51-A54) does also not change the internalization rate in CHO cells [Partial ablation or mutation of the retromer interaction motif in SorLA-CD caused endosomal accumulation of SorLA, altered APP sorting and increased APP processing , 63. Addargeting , 28. SorHO cells . HoweverHO cells . Taken tThe following primary antibodies were used: rabbit anti-dsRed (tdTomato) ; chicken anti-GFP (mVenus) ; mouse monoclonal anti-interleukin receptor 2 alpha  ; mouse monoclonal anti-Myc – 9E10 ; rabbit polyclonal anti-PICK1 ; mouse monoclonal anti-PICK1 ; rabbit anti-SorLA (SorLA-Vps10p domain)  [The construct of the SorLA-cd inserted in pGEX4T-1 (Amersham Pharmacia) has been described before . To gene2. Lipofectamine™ 2000 (Invitrogen) was used for transfection of cells. Generation of stable transfected cells was described before [CHO and COS7 cells were cultured in DMEM (Invitrogen) supplemented with 10% (v/v) fetal calf serum (FCS). SH-SY5Y cells were cultured in DMEM (Invitrogen) supplemented with 20% (v/v) fetal calf serum (FCS). Murine neuroblastoma N2a (Neuro-2a) cells were grown in Opti-MEM™ without Phenol Red (Invitrogen) supplemented with 5% (v/v) FCS. All cells were cultured at 37 °C and 5% COd before . Dissocid before . Animal Renilla luciferase module.Interaction of proteins was determined using a modification of the Checkmate mammalian two-hybrid system (Promega). Two vectors were used, pAct and pBind3-D. pAct (Promega) contains the herpes simplex virus VP16 activation domain followed by a multiple cloning site. pBind3-D  is a modN2a cells were seeded on 96-well plates and transfected in parallel with pAct, pBind3-D, peGFP (Clontech), and a pG5luc (Promega) expressing firefly luciferase under control of GAL4 . The nexGST or GST-fusion proteins were expressed in BL21(DE3) cells, and purified by using glutathione-sepharose beads according to the manufacturer’s recommendations (Amersham Pharmacia). Stable transfected CHO cells expressing PICK1-Myc were lysed in 150 mM NaCl, 1% Triton X-100, 20 mM Tris–HCl, 10 mM EDTA, pH 8.0 supplemented with protease inhibitors , cell debris sedimented for 20 min at 16.000 g and 10% of each supernatant was used as an expression control (input). Supernatants were incubated with 30 µl Pierce™ glutathione magnetic agarose beads (ThermoFisher Scientific) prebound with GST or GST-SorLA-cd for 2 h at 4 °C. Subsequently, protein complexes were subjected to immunoblotting.For pull down of endogenous proteins, brain homogenates were prepared from 2 mouse forebrains in 320 mM Sucrose, 4 mM Hepes, 2 mM MgCl, 2 mM CaCl, pH 7.5 supplemented with protease inhibitors  using a glass homogenizer (12 passes) (modified from ). HomogeCells were cultured on coverslips, fixed, permeabilized and stained as described before . To visu