Estimation & Study of Eclipta prostrata for the treatment of Various Disease

The use of plants in traditional medicines covers a wide range of therapeutic uses to treat the infection as well as many chronic diseases. Many people still rely on the traditional medicine and healthcare because of their wider cultural acceptance and affordability. The plant based bioactive compounds have been an important source of modern drugs discovery and development. Hence, the medicinal value of various plants should be explored with their pharmacological significance and potential application in different products. Eclipta prostrata is commonly known as False daisy or Ink plant in English and locally known as Bhringraj, Bhumiraj, Aali jhar, and Nash jhar in Nepali language. E. prostrata is a medium-sized, branched, annual herb-bearing white flower natively found in the tropical and subtropical regions of the world . It grows mostly in moist sites such as swamp edges, river or lake banks and edge of rice-fields and easily propagated and spread throughout China, India, Nepal, Brazil and other parts of the world . It is widely distributed in tropical and sub-tropical regions of Asia, Africa, and South America .Traditionally, it is used to treat different skin problems such as wounds, hair loss prevention, and dermatitis. The leaves are used to treat snakebite in India, China, and Brazil. The mixture of leaf juice and honey is used to cure catarrh in infants . The juice of E. prostrata is taken orally or applied locally to promote hair growth .Various research articles have been published regarding the chemical constituents  and biological activities of different plant parts of E. prostrata. Critical analysis of these published scientific studies would provide the detailed understanding about the potential use of E. prostrata as medicine, cosmetic, and other formulations along with highlighting the gaps in research. Hence, the main aim of this article was to collect the information about traditional uses, chemical constituents, and the biological activities of E. prostrata. Family  Asteraceae Eclipta Alba (Asteraceae) is an annual herbaceous plant, commonly known as false daisy. It is an erect or prostrate, much branched, roughly hairy, annual, rooting at the nodes; the leaves are opposite, sessile and lanceolate. It is also  known as Bhringaraj and Karisilakanni, which is found a common weed throughout India ascending up to 6000 ft. The specific Eclipta Alba means white which refers to  the colour of the flowers. Main active principles consist  of coumestans like wedelolactone, desmethylwedelolactone, furanocoumarins, oleanane & taraxastane glycosides. Ethnopharmacology : Eclipta Alba (L.) has   been used in various parts of tropical and sub-tropical regions like south America, Asia, Africa. There are three kinds or Eclipta Alba-the white-flowering, the yellow flowering, and the black-fruiting, but all three grow throughout India by marshes, rivers, and lakes or on the foothills of the Himalayas. It is an active ingredient of many herbal formulations prescribed for liver ailments and shows effect on liver cell generation. It is used as a tonic and diuretic in hepatic and spleen enlargement. It is also used in catarrhal jaundice and for skin diseases. The alcoholic extract of the plant has shown antiviral activity against Ranikhet disease virus. The plant is commonly used in hair oil all over India for healthy black and long hair . The fresh juice of leaves is used for increasing appetite, improving digestion and as a mild bowel regulator. It is commonly used in viral hepatitis to promote bile flow  and protect the parenchyma and popularly used to enhance memory and learning .The plant has a reputation as an antiageing agent in Ayurveda. It is used as a general tonic for debility. Externally it is used for inflammation, minor cuts and burns and the fresh leaf-juice is considered very effective in stopping bleeding. Leaf juice mixed with honey is also used for children with upper respiratory infections and also used in eye and ear infections. It is a source of coumestans type compounds used in phytopharmaceutical formulations of medicines prescribed for treatment of cirrhosis of the  liver and infectious hepatitis. It is widely used in India as a chologuague and deobstruent in hepatic enlargement, for jaundice and other ailments of the liver and gall bladder. The water extract of Eclipta prostrata (whole plant) exhibited the most potent inhibitory activity against HIV-1 integrase (HIV-1 IN). Vedic Guard, a polyherbal formulation is a synergistic combination of 16 medicinal plant extracts contains Eclipta Alba as a major ingredient. Charaka advises taking the juice of Eclipta Alba with honey to prevent the onset of senility, and its oil as the best medicated massage oils for rejuvenation therapie.        
       
           
                                                                                                

The plant reportedly contains a number of bioactive chemicals. The reported constituents are show. The structures of several phytoconstituents are shown in following structure,

Reported phytoconstituents of E. alba

The scientific information on E. prostrata was retrieved from various online bibliographic databases such as Science Direct, PubMed, Google scholar, SciFinder, etc. and books and proceedings. The articles with rigorous quality were selected for the review. Relevant articles published before June 2021 were collected using the key words Eclipta prostrata Eclipta alba, phytochemistry, traditional use, biological activity, pharmacological activity etc.

Ethnomedicinal Uses

This plant is widely used in different regions of India for the treatment of skin problems, hepatic problems such as jaundice, gastrointestinal problems, respiratory problems such as asthma, and other symptoms such as fever, hair loss and whitening of hair, cuts, and wounds, spleen enlargement, etc. . The leaf juice is used with honey to curecatarrh in infants, shoot juice and mustard oil is taken together for diarrhea and dysentery, and the whole plant is used for the treatment of symptoms related to hepatitis, itching,hemoptysis, bleeding, hematuria, diarrhea, and diphtheria . The leaves and shoots are used in preventing infection in wounds and its treatment in Nepal. Some ethnic groups in South American countries use it to treat snakebites . In Ayurveda, it is used for its revitalizing and anti-aging properties. Many ethnic groups of Bangladesh use it for the treatment of jaundice .The plant juice has been used to control, kill, and inhibit the growth of diseases carrying vectors such as mosquito . Additionally, it is also used to treat different types of symptoms such as acidity, alopecia  gingivitis, fever, body pain, asthma, bronchitis, burns, constipation, wounds, wrinkles, edema, pimples, and other skin diseases .

MATERIALS AND METHODS

Plant materials

Whole plants of E. prostrata were collected from green house Attached to the college campus, Kamaraj College of Engineering And Technology, Virudhunagar, and authenticated by Botanical Survey of India, Coimbotore. A voucher specimen was deposited in Our departmental laboratory. The whole plant was refluxed in Running tap water for 1 – 2 h. Leaves were detached and surface Sterilized by 0.1% (w/v) HgCl2 with two drops of Tween 80 for 2 min (Archana et al., 2004), followed by rinsing thrice with sterile distilled Water until all traces of sterilent are removed.

Bacterial cultures

Escherichia coli, Klebsiella pneumoniae, Shigella dysenteriae, Salmonella typhi, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus were purchased from IMTECH, Chandigar, India. Silica gel, organic solvents and other chemicals which were Used during this study were from Himedia, Merck and s.d. FineChemicals, Mumbai. Standard discs of tetracycline and Chloramphenicol purchased from Himedia.

Extraction

Surface sterilized leaves were subjected to solvent extraction by Various solvents like hexane, ethyl acetate (Et-oAc), ethanol (EtOH), and water in increasing order of polarity. Samples were extracted with each solvent one after another by soxhlet apparatus for About 24 h. Each solvent extract was distilled And concentrated in vacuole with addition of CaCl2. Lyophilized Aqueous fractions were further used to test for the antibacterial and Antioxidant properties.

Determination of antioxidants

The antioxidant activities of hexane, ethyl acetate, ethanol and Water extract of leaves of E. prostrata were determined by ferric Thiocynate method. 10 mg of each extract Was dissolved separately in 99.5% of ethanol and various Concentrations (50, 100, 250, 500 µg/mL) were prepared. A mixture Of a 2 mL of sample in 99.5% ethanol, 2.052 mL of 2.51% linoleic Acid in 99.5% ethanol, 4 mL of 0.05 M phosphate buffer (PH 7.0) And 1.948 mL of water was placed in a vial with a screw cap and Placed in an oven at 60°C in the dark. To 0.1 mL of this sample solution 9.7 mL of 75% ethanol and 0.1 mL of 30% ammonium thiocynate was added. After the addition of 0.1 mL of 2 x 10-2 M Ferrous chloride in 3.5% hydrochloric acid to the reaction mixture, The absorbance of the red color developed was measured in 3 min At 500 nm. The control and Standard were subjected to the same procedures as the sample, Except that for the control, only solvent was added, and for the Standard, sample was replaced with the same amount of  -Tocopherol (reference compound). The Inhibition of lipid peroxidation in percentage was calculated by Following equation: 

% Inhibition = 1 – (A1/A2)  100

Where A1 was the absorbance of the test sample and A2 was the Absorbance control reaction.

Antimicrobial test

Antibacterial assay of the crude extracts of hexane, ethyl acetate, Ethanol and water were performed by disc diffusion method using Muller Hinton agar. Wattman No. 1 filter discs were enriched with Approximately 50 µg of each solvent extract which was dissolved in n-hexane. Antibacterial activity was determined as diameter of the zone of inhibition (ZOI). ZOI measurements were made three times for each disc at different orientation and the average was recorded. Disc containing n-hexane alone was used as negative control and The standard tetracycline and chloramphenicol were used as Positive control at 30 µg concentration.

Determination of minimum inhibitory concentration (MIC)

The samples (hexane, ethyl acetate, ethanol and water extracts) Were dissolved in methanol. The solutions were individually added At different concentration from (0 (control) to 100 µg) to Muller Hinton agar and mixed well before being poured into sterile Petri Dishes. The bacterial cultures (10 µl) were taken from nutrient broth And added on the of inhibition (ZOI) of 10.24, 9.16, 9.14, 8.0, 7.60, 8.60 mm, respectively at 10 µg/mL and minimum inhibitory concentration (MIC) of 15, 25, 20, 1250, 1300, 1000 µg/mL respectively

Biological Activities

Due to the wide range of ethnomedicinal values and applications, several studies have Been performed regarding the biological activities of extracts and compounds obtained From E. prostrata using both In vitro and in vivo models . Some of them are discussed In detail in following sub-sections.

1. Antioxidant Activity

The antioxidant effects of E. prostrata were evaluated in Charles River Sprague-Dawley Rats. The extract at 50 mg/kg and 100 mg/kg dose significantly reduced the oxidative Biomarkers such as serum lipid peroxide, serum hydroxyl radical levels in .In another study, the in vitro antioxidant activity was evaluated based on the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay. An IC 50 value of extract was determined to be 45.68 µg/mL for the whole plant as compared to the IC50 of 3.26 µg/mL of standard ascorbic acid. When evaluated using hydrogen peroxide scavenging assay, the extract showed potent activity with the IC 50 values of 1.34 µg/mL as Compared to ascorbic acid (IC50: 1.03 µg/mL). The antioxidants present in the extract Of E. prostrata showed the reduction of ferricyanide complex (Fe+3) to ferrous form (Fe+2) In a dose-dependent manner. The highest reducing ability (75.59%) for the whole plant Of E. prostrata was reported at 250 µg/mL concentration. The IC 50 values for reducing Ability of the extract was 100 µg/mL [62]. The studies are mostly conducted using In vitro Methods and detailed mechanism is yet to be established.

2.Antimicrobial Activity

The butanol and water extract, at a concentration of 3 mg/disc inhibited the growth Of Bacillus cereus by 45% and 42%, respectively. The highest growth inhibition of 63% for Butanol extract and 54% for ethyl acetate fraction were reported against B. subtilis at the Concentrations of 3 mg/disc. The butanol extracted sample showed inhibition of 57%, 72%, And 89% at concentrations of 1, 2 and 3 mg/disc, respectively and that of ethyl acetate Extract showed the growth inhibitions of 40, 57, and 83%, at concentrations of 1, 2, and 3 mg/disc, respectively. Similarly, methanol extract inhibited the growth of Candida albicans By 38, 48, and 59% at concentrations of 1, 2, and 3 mg/disc, respectively. A coumestan derivative, wedelolactone (10 µg/mL), isolated from the plant showed Promising antibacterial properties against Staphylococcus epidermidis, Salmonella Typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, Shigella flexneri and Escherichia coli with the zone Of inhibition (ZOI) of 10.24, 9.16, 9.14, 8.0, 7.60, 8.60 mm, respectively at 10 µg/mL and minimum inhibitory concentration (MIC) of 15, 25, 20, 1250, 1300, 1000 µg/mL respectively .The percentage inhibition of 36.84, 38.94, and 47.36% was reported against Aspergillus Niger by 2 mg, 3 mg, and 4 mg of ethanolic extract of E. prostrata, respectively as compared To the inhibition of 64.21% by 100 µg of standard fluconazole. Similarly, the percentage Inhibitions of 20, and 28% were reported against Aspergillus ustus by 3 mg, and 4 mg of Ethanolic extract, respectively as compared to inhibition of 40% by 100 µg of standard Fluconazole. Similarly, against Aspergillus ochraceus, the percentage inhibition of 41.37, 44.82, And 51.52% in was reported for 2 mg, 3 mg, and 4 mg of ethanolic extract, respectively Compared to the inhibition of 37.93% by 100 µg of standard fluconazole . The alkaloids from the leaves were also studied for the antimicrobial properties against E. coli, P. aeruginosa, Shigella boydii, S. aureus and S. faecalis by agar-well diffusion and brothmicrodilution methods. The ZOI ranged from 9.8–16.5 mm in 500 µg/mL and MIC ranged From 42–89 µg/mL for the sample which was found to be comparable to the positive control Ciprofloxacin with MIC range of 0.8–1.3 µg/mL .

3. Hepatoprotective Activities

In vivo hepatoprotective activity was evaluated by Thirumalai et al. The aqueous Extract of leaves of E. prostrata was administered to carbon tetrachloride (CCl4 induced Hepatotoxicity in male albino rats. The extract (250 mg/kg b.w.) reduced the elevated Levels of all the biochemical parameters such as glutamic-oxaloacetic transaminase (GOT), Glutamine-pyruvate transaminase (GPT) and bilirubin. The chloroform extract of the roots and methanol extract of the leaves of E. prostrata Were investigated for hepatoprotective activity in CCl4-induced hepatotoxicity in male Albino rats by measuring the levels of lysosomal enzymes. The chloroform extract of roots Showed 47.96% reduction in the lysosomal enzyme whereas, methanol extract of leaves Showed a 72.8% reduction. The triterpenoid and alkaloid fractions obtained from the Methanol extract of showed the 78.78% and 60.65% reduction in lysosomal enzyme levels, Respectively. Similarly, triterpenoid saponin fraction and coumestan fractions obtained From the chloroform extract of roots reduced the lysosomal enzyme level by 52.41% and 75.6%, respectively [65]. The ethanolic extract (50%) obtained from the whole plant of E. prostrata was studied For its hepatoprotective effect. The study was conducted in rats against CCl4-induced Hepatic damage. The result revealed that E. prostrata extract significantly normalized the Biochemical parameters by counteracting the hepatic drug-metabolizing enzyme inhibition. The reduction of a biochemical parameter such as hepatic lysosomal acid phosphatase and Alkaline phosphatase by CCl4 was observed to be restored when treated with E. prostrata. This study shows that the hepatoprotective activity of this plant relies on the regulation Of the levels of hepatic microsomal drug-metabolizing enzymes. The echinocytes’ Acid and eclabasaponin II from the aerial parts showed antiproliferative activity in hepatic Stellate cells . The experiments were performed mostly in vivo using only rats. Its clinical significance is not well studied. Some studies lack positive and negative controls. Further, studies On human and animal models along with the isolation of active compounds from this plant May lead to the discovery of potent therapeutic agents.

4. Anti Hyperlipidemic Activities

Diacylglycerol acyltransferase (DGAT) is a key enzyme of biosynthesis in the final Step of the glycerol phosphate pathway. The triglycerides synthesized in excess causes Various symptoms such as type II diabetes mellitus, hypertriglyceridemia, and obesity. The Active polyacetylene constituents from the stem of E. prostrata was tested for the inhibition Of the DGAT enzyme. Kuraridine was used as positive control, which is known as DGA Inhibitor. A total of 8 isolates showed potent activity and IC50 values was of 74.4 ± 1.3 to101.1 ± 1.1 µM range while that of positive control was 10.4 ± 1.4 µM . The effect of methanol extract of E. prostrata on non-alcoholic fatty liver in rats was evaluated after inducing fatty liver via high-fat diets with cholesterol and cholic acids. The biochemical and histopathological analysis revealed that high dose treatment of E. prostrata (200 mg/kg and 300 mg/kg) exhibited significant improvement in lipid profile and liver function.

5. Cerebroprotective and Nervous System Related Activities

The hydroalcoholic extract of E. prostrata was subjected to the study of cerebroprotective function in Wister albino rats . The pre-treatments of the hydroalcoholic extract global cerebral ischemia model resulted in a great difference in ischemia control. E. prostrata extract (250 and 500 mg/kg) administration gradually improved the antioxidant enzyme levels, decreased brain edema, and altered some histopathological status in mice after bilateral cerebral artery occlusion. The aqueous extract obtained from leaves of E. prostrata at the dose of 100 and 200 mg/kg was evaluated for its potential application on transfer latency (TL) as a parameter of acquisition and retrieval learning in rats using an elevated plus-maze. The administration of extract was reported to significantly improve the retrieval memory .

   The acetylcholine formation in the brain and oxidative stress inhibition in the brain and serum of rats were accessed before and after feeding the experimental diet. The rats were fed with 25, 50,100 mg/kg of butanol fraction of the aqueous extract of E. prostrata. The acetylcholine level was increased by 9.6–12.1% in 50 mg/kg and 100 mg/kg fed group as compared to control. Monoamine oxidase-B activity and superoxide radical levels were decreased by 10.5% and 9.4%, respectively in the 100 mg/kg treated group. The in vivo anti-epileptic activities were studied by Tambe et al. in mice . This study isolated the luteolin as a major constituent from the leaves of E. prostrata which was evaluated for its anticonvulsant and anti-epileptic activities. The study found that luteolin exhibited anticonvulsant activities and also ameliorated the oxidative level in the mice induced by kindling.

6. Anti-Diabetic/Anti-Hyperglycemic Activities

The in vitro ?-amylase inhibition activity of methanol extract of the whole plant of

E. prostrata was evaluated. The result revealed mild potency in ?-amylase inhibition indicating the potential anti-diabetic property with the IC50 value of 322.138 ± 0.025 µg/mL [72]. In another experiment, the rats were injected with streptozotocin in the peritoneum at a dosage of 70 mg/kg of body weight to induce diabetics. With the administration of wedelolactone from E. prostrata to diabetic rats, HbA1c (%) level was recovered from 10.3 ± 0.72 in the untreated diabetic rats to 7.2 ± 0.52 in wedelolactone treated diabetic group. The change in a biochemical parameter such as an increase in urea and creatinine in the streptozotocin treated group was declined towards normal by treating with wedelolactone. The c-peptide and insulin secreted from ?-cells were investigated to confirm the treatment by wedelolactone and these hepatic-parameters were shifted towards the normal values after 28 days of treatment of wedelolactone. The ethanolic extract of E. prostrata showed inhibition of ?-glucosidase in a dosedependent manner. About 85% inhibition was observed at 100 µg/mL concentration while that of standard acarbose showed around 56% inhibitory at the same concentration. The IC50 value of the extract was 54 µg/mL. The aldose reductase activity was also found to be inhibited in a dose-dependent manner. The maximum inhibition was observed at the concentration of 10 µg/mL at which the enzyme activity was decreased by 88.6%. The IC50 values of the extract were calculated to be 4.5 µg/mL [74]. Many polyherbal formulations include E. prostrata as an essential ingredient. It is reported to act upon the pancreas via restoration and regeneration of ?-cell and to possess antidiabetic activity . The antidiabetic properties of E. prostrata in alloxan-induced diabetic rats were evaluated using the leaf suspension of the plant (2 & 4 g/kg) orally. The extract significantly normalized the biochemical parameters altered by diabetes. The activity of liver hexokinase was increased; blood glucose level and glycosylated hemoglobin were reduced due to the reduced activity of glucose-6 phosphatase and fructose-1,6-bisphosphatase which revealed the anti-hyperglycemic activity of E. prostrata in the rats . However, the mechanism on the chemical model related to the antidiabetic properties has not been well studied.

7. Anticancer Activities

The methanol extract of E. prostrata was administered orally at the dose of 250 and 500 mg/kg to Ehrlich ascites carcinoma (EAC) bearing mice and it was found to increase lifespan. It also decreased the viable cell count and tumor volume of the tumor-bearing mice when compared to that of control. As compared to EAC control, E. prostrata extract restored the hematological parameters such as red blood cells (RBC) count and hemoglobin content. In the treated group, the percentage of lymphocytes was increased with a decreased level of neutrophils . The in vitro and in vivo tumor growth inhibition of breast cancer cells by the chloroform fraction of methanol extract of E. prostrata was reported by Arya et al.  which resulted the marked inhibition of the breast tumor growth in vitro and in vivo by selective regulation of Hsp60 cell. The extract specifically activated the apoptotic pathway by the process of disruptions of mitochondrial membrane potential by upregulating and downregulating the Hsp60 and anti-apoptotic protein XIAP, respectively. The extract was also found to mitigate tumor-induced hepato-renal toxicity. Further, the LC-MS approach identified luteolin as a major contributor to the anti-cancer activities. The antitumor activities of terthiophenes isolated from the n-hexane fraction of E. prostrata were evaluated against the endometrial cancer cells (Hec1A and Ishikawa cells) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Among the five terthiophenes isolated, ?-terthienylmethanol was found to be most potent to inhibit the Hec1 (IC50 = 0.38) and Ischikawa (IC50 = 0.35) cancer cells .The cytotoxic activities of various compounds isolated from aerial parts of E. prostrata were evaluated against human ovarian cancer cell lines SKOV3. Cytotoxicity was assessed by MTT assay, and the IC50 values of more than ten compounds were less than 100 µM and that of positive control cisplatin was 11.25 ± 0.27 µM .The antiproliferative potential of ethanolic extract of E. prostrata was evaluated using MTT assay in which the extract inhibited the cell proliferation in a dose-dependent manner. The IC50 value for MTT assay was calculated from the growth curve and was found to be 22.1 ± 2.9 g/mL for HepG2, 25.3 ± 3.6 g/mL for A498, and 50.2 ± 8.7 g/mL for C6 cell lines. This result showed that extract was much effective for HepG2 cells. When treated with different doses of the extract resulted in a decrease in cell density which was supported by the MTT assay. The cells in all three cell lines were found to be detached, round, and floating at higher concentrations of extract . However, the effects on the biochemical pathway of healthy cells have not been well studied yet.

8. Hair Growth Promoting Activity

The dorsal area was shaved and the effect on growth of hair was observed in Wister rats by Mondal et al. to evaluate hair growth promoting activity .The time for the hair growth initiation was noted in days and the time required to complete the hair growth was 19.01 ± 0.51 days for ethanolic extract of the leaves of E. prostrata treated group compared to the time taken for 2% minoxidil (standard) treated group (16.05 ± 0.41 days). The hair growth in the extract-treated group observed at the 10th, 20th, and 30th day was 8.91 ± 0.03 mm, 16.01 ± 0.01 mm, and 21.08 ± 0.03 mm, respectively as compared to the length of hair increased in 2% minoxidil (standard) treated group as 9.23 ± 0.01 mm, 17.63 ± 0.02 mm and 22.13 ± 0.04 mm, respectively. Both the extract and standard treated groups showed a significant increase in as compared to the control group. The effects of petroleum ether extract of E. prostrata on stimulation of the hair coverage area of the nude mice were evaluated after applying specific concentrations of petroleum Ether extract vs. the vehicle and or 2% minoxidil.  A score of 0 to 8 was given for each Mouse to estimate the effects of petroleum ether extract on the hair coverage area of the Mice in all treatment groups. From day 8 the mice treated with petroleum ether extract of E. prostrata showed maximum hair growth than in the mice of other groups and gradually Covered the maximum area of the body on day 16. But rapid hair loss was observed in The minoxidil and other treatment groups in the case of the nude mice. In terms of hair Density, the mice treated with petroleum ether extract exhibited a significant increase in Hair density compared to the other groups on day 8 and 16. Although minoxidil had a Significant effect on sustaining hair density on day 8, progressive hair loss occurred and Hair density also decreased on day 16 . The hair growth cycle was found to be significantly affected by minoxidil and petroleum Ether extract of E. prostrata treatment. In the case of control, one to two hair follicles were In the catagenic phase, while most were in the telogen phase. A similar scenario was Observed in the group treated by ethanolic extract with the absence of anagen hair follicle. The reverse scenario was observed in minoxidil and petroleum ether extract treated group; Where most hair follicles were in anagenic phase, a few hair follicles in catagen phase and Almost no hair follicles were in telogen phase. In the control group, there were low anagenic Hair follicles, but in the case of the 2% and 5% petroleum ether extract group, it was about 68 ± 1.2% and 70 ± 1.6% anagenic hair follicles, respectively. The major contributors were Wedelolactone and ?- sitosterol in petroleum ether extract, which were responsible for hair Growth promotion . The study on hair growth promotion by a polyherbal formulation containing E. prostrata Was reported by Roy et al. . The time taken for complete hair growth was 18 d in the Group receiving oil of 10% E. prostrata, 10% hibiscus, and 5% jatamasi (DF3) and 22 d in The group receiving 10% E. prostrata, 5% hibiscus and 10% jatamasi (DF2). On comparing The activity of DF3 and minoxidil, DF3 hair oil formulation showed a better result of hair Growth. Mean hair length was 4.6 mm and 3.6 mm in DF3 and DF2 groups, respectively. The study revealed the fact that DF3 formulation resulted in significant increment in the Number of hair follicles in the anagen phase of the hair growth cycle. The percentage of The population of anagen follicle was 67 in the standard group, while in DF3 and DF2 Formulations, it was 82 and 65, respectively. The results revealed that DF3 formulation had More potent to hair growth activity [83]. However, the role of E. prostrata in the treatment Of hair fall caused by other reasons such as diseases, aging, and genetics are not clear yet.

9. Immunomodulatory Activities

The immunostimulatory effects of E. prostrata in tilapia fish (Oreochromis Mozambicans) Was studied. A diet with 0.01, 0.1, and 1% of the aqueous extract of E. prostrata were fed To fish, and after successive weeks, various nonspecific humoral responses (complement, Antiprotease, and lysozyme), cellular responses (reactive oxygen and nitrogen production, Myeloperoxidase content) and disease resistance were observed against the activity of Common pathogen of fish and human, Aeromonas hydrophila. After feeding aqueous extract For 1, 2, or 3 weeks, the activity of lysozyme was increased significantly. After 1 week Of aqueous extract supplement, the enhancement of reactive oxygen species production And myeloperoxidase content was observed. The mortality rate in fish had decreased Significantly when fed with the extract . The wedelolactone obtained from the methanol extract of the whole plant of E. prostrata Was reported to show immunomodulatory responses in mice at different dose ranges from 100 to 500 mg/kg. Various parameters such as carbon clearance, antibody titer, and Cyclophosphamide immunosuppression were accessed, which showed the significant Increase in the phagocytic index and antibody titer which resulted in a significant ratio of The phagocytic index and white blood cells (WBC) count

10. Others Activities

The study using In vitro assay suggested that wedelolactone from E. prostrata extract Exhibited anti-hepatitis C virus (HCV) activity. It was reported that the mechanism was Due to the inhibition of HCV replicase activity in the cell culture systems treated with Wedelolactone .E. prostrata was reported to exhibit active inhibition activity against Bothrops0 jararacussu Venom. The root extract was reported to have inhibition of the phospholipase A2 activity Nearly by 50%. The highest result was obtained for the extract of aerial part as compared To other extracts, which showed approximately 70% inhibition of the phospholipase A2 Activity . Echniocystic acid, the triterpene component of E. prostrata was found to be effective In treatment of ovariectomy-induced osteoporosis in rats. Administration of 5–15 mg/kg Per day for 12 weeks was reported to prevent the level of stress and Young’s modulus of The femur. The compound also restored the bone biomarkers level such as osteocalcin, Alkaline phosphatase, deoxypyridinoline, phosphorus, and urinary calcium. Treatment From E. prostrata also prevented the altercation of the bone mineral density, improved Trabecular architecture, trabecular number, and trabecular thickness .The methanol extract of leaves of E. prostrata was reported to show nephroprotective Activity in gentamycin-induced nephrotoxicity in rats. The activity of the extract was Evaluated for its ability to decrease the gentamicin-induced elevations of biochemical Parameter such as serum creatinine and histological changes in renal tissues. It was Reported that the extract of could significantly act as a nephroprotective agent against Gentamicin toxicity comparable to quercetin . Similar studies were made on doxorubicin Hydrochloride-induced nephrotic syndrome on mice. The significant improvement on Biochemical parameter such as urine protein, triglyceride etc. was seen on the E. prostrata Treated group. The pathological changes in kidney also supported the fact . Another Study revealed that the major constituents of E. prostrata i.e., wedelolactone could inhibit The abnormal proliferation of human renal mesangial cells (HRMCs) due to inflammation Of renal tissues via the regulation of NF-?B signaling pathway .

Table 1: Parts containing chemical constituents of Eclipta prostrata

Organoleptic, macroscopic and microscopic Analysis

Coarsely powdered sample of whole plant was Evaluated for color, odor, taste, texture, etc. The Organoleptic characters of the sample were evaluated Based on the method described by Morphology and anatomy of both vegetative And reproductive parts including the stem, leaf, Flower, fruit and seed were examined microscopically. Powder microscopy Powdered samples of the whole plant of E. prostrata  Were examined under the microscope for their  Characteristic features following WHO guidelines.

Physico-chemical analysis

The parameters which were studied are the moisture  Content, water and ethanol extractable matters and Total, acid insoluble and water-soluble ash values of  The powder of whole plant of E. prostrata. Presence or absence of heavy metals such as Cadmium (Cd), lead (Pb), arsenic (As), and mercury (Hg) were detected using an Inductively Coupled Plasma – Mass Spectrometer (ICP-MS).

Preliminary phytochemical screening

The coarse powder was extracted into hexane, Dichloromethane, ethyl acetate, methanol and water Using continuous Soxhlet extraction in accordance With their increasing polarity. The ratio of plant Material to solvent was 1:5. Qualitative Phytochemical screening of different extracts was Carried out according to the methods described by Farnsworth (1966) and Evans (2009).

Materials and Methods

Collection of plants and authentication

   Eclipta prostrata L. whole plant was procured From Thrissur and authenticated at Botanical Survey of India, Coimbatore. The leaves were Collected; shade dried and pulverized using an Electrical pulveriser. About 50 g powder was Taken and extracted with methanol using Accelerated solvent extractor (Dionex ASE150, Thermoscientific). The methanolic Extract was then concentrated using a rotary Vacuum evaporator under reduced pressure And Temperature And Stored Under Refrigeration (4ºC) until further use.

Phytochemical screening

   The leaves were tested for the presence of Various active chemical constituents namely Steroids, Alkaloids, Tannins,Phenolic Compounds, Flavonoids, Glycosides, Diterpenes, Triterpenes And Saponins. Fourier Transform Infrared (FTIR) Spectroscopy Functional groups present in E. prostrata Leaves were identified using Fourier transform Infrared (FTIR) spectroscopy. About 2.0 mg of E. Prostrata leaves powder and 298 mg of dry Fine powder of potassium bromide (KBr) Were mixed well using mortar and pestle. The KBr- sample mixture was transferred to an Evacuable die that has a barrel diameter of 13 Mm and the die was pressed at around 8 to 10 Tons for 1-2 min in a hydraulic hand press. Re-crystallization of the KBr results in a clear Transparent disk about one millimetre thick And the infrared spectrum was recorded in the Scan range from 4000 cm–1 To 400 cm–1 Of FTIR spectrophotometer with a resolution of 0.5 cm-. The structurally related compound Were identified through Fluka library supplied By Perkin-Elmer.

GC-MS analysis The GC-MS analysis of the extract was Carried out at Kerala Forest Research Institute, Peechi, Thrissur using Shimadzu GCMS Model Number: QP2010S. It was Performed as per the protocol described by Anand Et al.,  (2014) With Minor Modifications. The compounds were separated on Rxi-5Sil MS capillary column (30 m × 0.25 mm; i.e., 0.25 ?m film). The sample dissolved in Methanol, filtered in 0.22-micron syringe Filter, was used for analysis. The column oven Temperature was programmed from an initial Temperature of 80ºC (4 min), then temperature Raised to 280ºC at the rate of 5ºC min-1, Finally 280ºC was maintained isothermally With a final time of 6 min. The injection Temperature and ion source temperature were 260ºC and 200ºC, respectively. Helium (99.999%) data were obtained by Collecting the full-scan mass spectra within The scan range 50–500 amu.

Mechanism of action of Eclipta prostrata