Location:

It occupies a substantial part of the abdominal cavity. It lies under the diaphragram. It occupies most of the right hypochondrium, part of the epigastrum and extending into the left hypochondria region.

Loops and cells:

The liver is divided into primary loops. The two primary cells of the liver are the hepatocytes and kupffer cells (Sherwin and Sobenes, 1996). Hepatocytes constitute about 80% of the liver by volume and 60% of the liver by number. They form a contentious system or murale of hepatic laminae. Hepatocytes are polyhedral with 5-12 sides. Their nuclei are euchromatic and polyploidy. Their cytoplasm displays much granular and agranular endoplasmic reticulum, mitochondria lysosomes, golgi bodies. They contain characteristic type of cytokeratin filaments, Hepatocytes mediate many activities.

Hepatocytes:

Hepatocytes comprise the bulk of the organ and play a central role in drug metabolism. Hepatocytes are the chief functional cells of the liver and perform an astonishing number of metabolic, endocrine and secretary functions. Roughly, 80% of the liver is contributed by hepatocytes. In three dimensional view; hepatocytes are arranged in plates that anastomose with one another. The cells are polygonal in shape and their sides can be in contact either with sinusoid (sinusoidal face) or neighboring hepatocytes (lateral face). A portion of the lateral faces of hepatocytes is modified to form the bile canaliculi. Microvilli are present abundantly on the sinusoidal face and project sparsely into bile canaliculi. Typical hepatocyte is in contact with the hepatic blood, other hepatocytes and bilary capillary. There are always large amounts of endoplasmic reticulum with many mitochondria and micro bodies in each hepatocyte.

HERBAL REMEDY FOR LIVER DISORDRES:

The effective alternative treatment of the stated condition can be obtained with herbal products that are known to cure more than one disease or disorder. Plants have been used for medicines because they have fitted the immediate personal need; they are accessible and inexpensive. Herbal products have the reputation of long history of use and better patient tolerance. Traditional system of medicine employing mainly plant based drugs are always appreciated in pharmaceutical research as the major resource for new medicines and a growing body of medical literature supports the clinical efficacy of herbal plants. The literature survey reveals that much work was not done on the plants;

1) Eclipta alba - whole plant

2) Centella asiatica - leaf

3) Tinospora cordifolia - root and bark

4) Phyllanthus amarus - root and bark

5) Ricinus communis - leaf

Based on the careful study of above points, it was felt, it is worth to do extensive study on these plants. Hence, the present study deals with the hepatoprotective study of the above plants and definitely this work will through some light for the innovation in the field of pharmacy.

SELECTION OF DRUGS:-

The following herbal drugs are selected for the study as they have the reputation of being used in folk/traditional medicine to treat hepatic disorders and/or their hepatoprotective potential has been scientifically reported. The criteria for selection include the antioxidant potential of the herbs also. The selected five drugs have been shown to possess free radical scavenging activity

Stem of Tinospora cordifolia.

Leaves of Ricinus communis.

Whole plant of Eclipta alba.

Stem of Phyllanthus amarus.

Leaves of Centella asiatica.

MATERIALS AND METHODS:

Chemicals:

1, 1-Diphenyl-2-picryl hydrazyl (DPPH) and 2,2’-Azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) were obtained from Sigma Aldrich Co., St. Louis, USA. Rutin from Acros Organics., New Jersey, USA. Naphthyl Ethylene Diamine Dihydrochloride (NEDD) from Roch – Light Ltd., Suffolk, UK. Ascorbic acid, Nitro blue tetrazolium (NBT) and Butylated hydroxy anisole (BHA) from SD Fine Chemicals Ltd., Mumbai, India. Sodium Nitroprusside from Ranbaxy Laboratories Ltd., Mohali, India. Sulphanilic acids from E-Merck (India) Ltd., Mumbai, India were used. All chemicals used were of analytical grade.

Estimation kit:

· ALAT: E-Coline kits Merck (India) Ltd., Mumbai.

· ASAT: E-Coline kits Merck (India) Ltd., Mumbai

· ALP : E-Coline kits Merck (India) Ltd., Mumbai.

Instruments

a) Spectrophotometer (UV-VIS) : Perkin Elmer, USA

b) HPTLC Instrument : CAMAG LINOMAT-IV and CAMAG TLC scanner 3

Cell Lines:

The cell line was obtained from National Centre for Cell Sciences, Pune, India.

BRL-3A

Source :Rat

Morphology :myoblast

Origin :Liver

Serum :New born calf serum

Plant profile:

PHARMACOGNOSTICAL STUDIES:-

A] Centella asiatica Linn²

Phytochemistry:

Leaves contain highly variable triterpenoid saponins, including asiaticoside, oxyasiaticoside, madecasoside (Madagascar chemotype), centelloside (Sri Lankan chemotype), brahmoside, brahminoside, thankunoside, isothankunoside and related sapogenins from various chemotypes. It also contains triterpenoid acids viz.asiatic acid, madecassic acid, brahmic acid, isobrahmic acid and betulic acid etc ^3-10.

Active principles:

Asiaticosides, Madecassoside, Asiatic acid, Madecassic acid

METHODS:

1) Test for flavanoids:

Shinoda test:

To the extract, magnesium turnings and then concentrated hydrochloric acid is added. Red colour is produced.

Estimation of total phenolic content:⁴¹

Phenols comprise the largest group of plant secondary metabolites. They range from simple structures with only one benzene ring to larger molecules such as tannins, anthraquinones, flavonoids and coumarins. They are defined as compounds that have at least one hydroxyl group attached to a benzene ring. Phenolic compounds are commonly found in both edible and inedible plants and they have been reported to have multiple biological effects, including antioxidant activity.

Total phenol was determined in powder crude drugs, extracts and beverages by using the Folin-Ciocalteu method. This test is based on the oxidation of phenolic groups with phosphomolybdic and phosphotungstic acids. After oxidation a green blue complex is measurable at 750 nm. The total phenol content of a tested material is being related to the antioxidant activity shown by it.

Chemicals and Regents:

1. Folin-Ciocalteu Reagent:

Folin-Ciocalteu reagent was diluted (1:10) with distilled water and used.

2. Sodium carbonate:

202.5 g of sodium carbonate (Na₂CO₃10 H₂O) was dissolved in 1 liter of distilled water and used.

3.Methanol:

Distilled

Preparation of Test Solutions:

1. 10 mg each of these extracts were separately dissolved in 10 ml of methanol to get 1mg/ml solution. These solutions were diluted with methanol to obtain lower dilutions.

Preparation of Standard Solution:

1. Gallic acid:

100 mg of gallic acid monohydrate was dissolved in 100 ml of distilled water to get 1000 mg/ml solution. It was serially diluted with distilled water to obtain solutions ranging from 25 mg/ml to 250 mg/ml.

Procedure:

400 ml of the extracts (1 mg/ml to 0.5 mg/ml) were separately mixed with 2 ml of Folin-Ciocalteu reagent and 1.6 ml of sodium carbonate solution. After shaking, it was kept for 2 h reaction time. The absorbance was measured at 750 nm (Shimadzu UV-160 A Spectrophotometer, Shimadzu Corporation, Japan). Using gallic acid monohydrate, standard curve was prepared and linearity was obtained in the range of 2.5 to 25 mg/ml. Using the standard curve the total phenol content of extracts was obtained . The total phenol content was expressed as gallic acid equivalent in mg/g or % w/w of the extracts.

Estimation of total flavonol content:⁴²

Flavonoids are water-soluble polyphenolic compounds, which are extremely common and wide spread in the plant kingdom as their glycosides. The word ‘flavonoid’ is derived from the Latin word “flavus” meaning yellow and many flavonoids are indeed yellow in color. It consists of a single benzene ring joined to a benzo-gamma-pyrone structure. They are able to complex metal ions, acts as antioxidants and bind to proteins such as enzymes and structural proteins.

The different classes within the group are distinguished by additional oxygen containing hetrocyclic rings and hydroxyl groups. These include the catechins, leucoanthocyanidins, flavanones, flavones, anthocyanidins, flavonols, chalcones, aurones and isoflavones.

Total flavonol was determined by aluminum chloride colorimetric method. The principle of this method is that aluminum chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5 hydroxyl group of flavones and flavonols. In addition, aluminum chloride forms acid labile complexes with the ortho-dihydroxyl groups in the A or B ring of flavanoids.

Chemicals and Reagents:

1. Aluminum chloride (10%):

10 g of aluminum chloride was dissolved in 100 ml of distilled water, filtered and used.

2. Potassium acetate (1 M):

98.1 g of potassium acetate was dissolved in 1 liter of distilled water and used.

3. Methanol:

Distilled

Preparation of Test Solutions

1. 100 mg each of the extracts were dissolved in 10 ml of methanol to get 10 mg/ml solutions.

Preparation of Standard Solution

1. Rutin:

10 mg of rutin was dissolved in 100 ml of methanol to get 100 µg/ml solutions. It was serially diluted with methanol to obtain solutions ranging from 10 µg/ml to 100 µg/ml.

2. Procedure:

0.5 ml of the extracts was separately mixed with 1.5 ml methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm with a Shimadzu UV-160A Spectrophotometer (Shimadzu Corporation, Japan). Using rutin, standard curve was prepared and linearity was obtained in the range of 1-10 µg/ml. Using the standard curve the total flavonol content of extracts was obtained. The total flavonol content was expressed as rutin equivalent in mg/g or % w/w of the extracts.

High performance thin layer chromatography (HPTLC):

HPTLC is a modern chromatographic technique in which the principle of TLC is sophisticated and automated by which the samples are accurately and precisely estimated which can be utilized for both qualitative and quantative purpose.

Ø Characteristic of HPTLC layer:

1. Thinner 0.2 mm

2. Smaller grain size (7 µm) and closer distribution.

3. Better separation performance over a 50% shorter optimum separation distance (50 mm).

4. Optical properties ensure a better signal to noise ratio in densitometric evaluation.

5. On HPTLC layers, Chromatography takes place in the fastest capillary flow range of the mobile phase.

Ø Merits of HPTLC over conventional TLC:

1. Faster separation

2. Reduced diffusion, hence improved separation efficiency.

3. Detection (Determination) limits lower by a factor of 10-15.

Ø HPTLC necessitates the following:

1. Appropriate dimensioning of the applied volume.

2. High quality application

3. Appropriate developing equipment and procedures.

General procedures:

Ø Preparation of sample:

Preparation of sample and standard is an important tool in HPTLC analysis. Sample should be prepared in a solution form with suitable solvents in proper concentration. The selection of solvents depends on the type of solvent used for extraction.

Ø Application of sample:

Commercially available precoated HPTLC plates (Merck) can be used for the study. The solutions of various concentrations should be applied on the respective HPTLC plates using Linomat IV applicator. The plates were dried after application.

Ø Development of plates:

Plates should be developed in a suitable solvent system to the premarked distance. These plates should be removed from the chamber, dried and scanned.

Ø Detection:

The developed plates should be observed under daylight and UV light for the detection of constituents.

Ø Densitometric scanning:

The developed plates should be scanned at a suitable wavelength for the quantative and qualitative analysis. Peak areas and peak heights should be recorded from which unknown concentration of the samples can be determined.

Fingerprint of extracts of Centella asiatica, Eclipta Alba, Phyllanthus amarus, Ricinus communis and Tinospora cordifolia. The extracts of Centella asiatica, Eclipta Alba, Phyllanthus amarus, Ricinus communis and Tinospora cordifolia were subjected to HPTLC and the peaks obtained were identified.

Ø Preparation of sample:

Accurately weighed about 10 mg each of crude extract of Centella asiatica, Eclipta Alba, Phyllanthus amarus, Ricinus communis and Tinospora cordifolia and was dissolved in 10 ml of methanol in a 50 ml beaker with stirring for few minutes. The solution was filtered through Whatman filter paper and transferred into 10 ml standard flask. The sample solution thus prepared was used for HPTLC analysis.

Ø Preparation of standard.

Accurately weighed about 1 mg of standard drugs like Phyllanthin and was dissolved in 5 ml of methanol in a 50 ml beaker with stirring for few minutes. The solution was filtered through whatman filter paper and the solution was made to 10 ml in 10 ml standard flask, the same procedure was followed for the preparation of other standard extracts viz. Hypophyllanthin, Asiaticoside and berberine.

Table no. 2. The standard solution thus prepared was used for HPTLC analysis.

S. no.	Standard markers used	Extracts
1.	Phyllanthin and ypophyllanthin	Phyllanthus amarus
2.	Asiaticoside	Centella asiatica
3.	Berberine	Tinospora cordifolia

Ø Application of sample:

Various concentrations of extracts of Centella asiatica, Eclipta alba, Phyllanthus amarus, Ricinus communis and Tinospora cordifolia in different concentration were applied on the HPTLC plats using Linomat IV applicatior.

Ø Solvent system:

All the solvents used for HPTLC were of HPLC grade.

The solvent system used for Centella asiatica, Eclipta alba, and Phyllanthus amarus, was

acetonitrile : methanol

1 : 1

The solvent system used for Ricinus communis was

acetonitrile : methanol

7 : 3

The solvent system used for Tinospora cordifolia was

n-propanol : formic acid : water

90 : 01 : 09

Detection:

The plate developed was dried and the plates were observed under UV light and scanned at the wavelengths of 200 nm, 254 nm and 356 nm. The finger printing of crude extracts of Centella asiatica, Eclipta Alba, Phyllanthus amarus, Ricinus communis and Tinospora cordifolia are observed and scanned by Linomat scanner to get spectra.

In vitro hepatoprotective screening:

In vitro hepatoprotective activity using established live cell culture and estimation of bio-chemical parameters.⁴³

1. The mono layer of the cell culture (BRL-3A) was trypsinized and the cell count was adjusted to 1.0×10⁵cells/ ml using medium containing 10% calf serum.

2. To each well of the 96 well micro titer plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was added. The plates were incubated at 37 ° C in 5% CO₂ atmosphere in a carbon dioxide incubator

3. After 24 hours, when partial monolayer was formed, the supernatant was flicked off, washed the monolayer once.

4. Then all the wells, except cell control wells were intoxicated with 50 ml of 1% carbon tetrachloride, followed by addition of 50 ml of different concentrations of test drugs. The cell control wells received 100 ml of maintenance medium. Then the plates were incubated at 37° C in 5% co₂atmosphere for 24 hours.

5. Toxicant control with CCl ₄ intoxicated wells without drug treatment, and standard (Liv-52) were also maintained.

6. After incubation, toxicant and drug/standard treated cell supernatants were pooled and collected separately in eppendorff tubes and centrifuged at 4,000 rpm for 15 min.

Supernatants were collected and following enzymes levels were estimated.

v ASAT

v ALAT

v ALP

RESULTS:

PHYTOCHEMICAL STUDIES:

Table No: 03. Phytochemical Studies on Centella asiatica, Eclipta alba, Phyllanthus amarus, Ricinus communis and Tinospora cordifolia.

S. No.

TESTS

INFERENCE

amarus

C. asiatica

R. communis

E. alba

T. cordifolia

Flavones and Flavonoids

(+) Present, (-) Negative

a)Total phenol estimation:

Among all the five plants extracts, the hydroalcoholic extract of Ricinus communis has shown high total phenol content, 2.51 % (Table 4). The other four extracts showed total phenol content in the range of 0.63 to 1.58%.

Table. No: 04. Total phenol estimation of five plant extracts

S. No.	Plant extracts	% phenol content
1	Phyllanthus amarus	0.64
2	Centella asiatica	0.63
3	Ricinus communis	2.51
4	Tinospora cordifolia	0.91
5	Eclipta alba	1.58

b) Total flavonoid estimation of plant extracts:

Among all the five extracts, the hydroalcoholic extract of centella asiatica has shown high total flavonoid content, 42 mg/g the other four extracts showed total phenol content in the range of 1.8 to 4.6 mg/g.

Table. No: 05. Total flavonol estimation of five plant extracts.

S. No	Plant extract	Total flavonoid content (mg/g)
1	Phyllanthus amarus	4.6
2	Centella asiatica	42
3	Ricinus communis	1.8
4	Tinospora cordifolia	2.3
5	Eclipta alba	2.1

SUMMARY AND CONCLUSION:

Hydroalcoholic extracts of different parts of five different plants viz. Phyllanthus amarus, Centella asiatica, Ricinus communis, Tinospora cordifolia, and Eclipta alba were evaluated for hepatoprotective activity by in-vitro methods.

In vitro studies were carried out using BRL-3A cell culture. In BRL-3A, plant extract concentrations ranging from 62.5 mg/ml – 250 mg/ml were studied, using 1% CCl₄as toxicant. All the extracts showed considerable protectivity against CCI₄ induced toxicity in BRL-3A cells. All the biochemical parameters were estimated and compared with that of the control. In this study an innovator product (Liv-52, Himalaya) has been used along with the tests extracts. This innovator product showed very good restoration of enzyme levels to normal. Further hydroalcoholic extracts of different parts of five different plants viz. Phyllanthus amarus, Centella asiatica, Ricinus communis, Tinospora cordifolia, and Eclipta alba can be screened for hepatoprotective activity, which will be worthwhile in the estimation of the exact dose of the plant extract for its hepatoprotective activity. A large number of plants have shown potent hepatoprotective and antioxidant activities. Hence, we were interested to screen the extracts of these plants for their Hepatoprotective and antioxidant potentials using standard in-vitro models. Phytochemical studies on these plants showed the presence of flavonoids, phenolic compounds .

Among all the five plants extracts, the hydroalcoholic extract of Ricinus communis has shown high total phenol content, 2.51 % (Table 02). The other three extracts showed total phenol content in the range of 0.63 to 1.58%.

Total flavonol content of all five plants extracts were estimated. Among all the five extracts, the hydroalcoholic extract of Centella asiatica has shown high total flavonoid content, 42 mg/g the other four extracts showed total phenol content in the range of 1.8 to 4.6 mg/g.

Finger printing and quantitative estimation of extracts by HPTLC analysis showed that Centella asiatica contain asiaticoside, Tinospora cordifolia (Root and stem) extract contain berberine and Phyllanthus amarus contain hypophyllanthin and phyllanthin as active constituents, which have proven their role as antioxidant and hepatoprotective.

In Vitro Hepatoprotective activity of plant extract on liver cell culture (BRL-3A):

Established hepatocytes challenged with CCl₄(1% or 10 µg/ml) treated with different concentration of extracts and incubated for 24 hours at 37 ⁰C. Effect of the extract on the biochemical parameters viz., ASAT, ALAT and ALP is estimated from the supernatant and tabulated in table no. 18.

The groups of wells to which toxicant was added showed a significant deviation in the enzyme levels compared to the normal. An increase was observed in the levels of ASAT, ALAT and ALP.

All the parameters viz., ASAT, ALAT and ALP was almost restored to normal on exposure to 250 mg/ml, 125 mg/ml of hydroalcoholic extract of five different plants.

All the parameters viz., ASAT, ALAT and ALP levels were almost restored to normal on exposure to 250 mg/ml and 125 mg/ml of methanolic extract of hydroalcoholic extract of five different plants.

It is observed that all the biochemical parameter was restored to normal on exposure to 1% CCl₄ of innovator product. The innovator product was able to restore all the biochemical parameters tested about to normal at concentration of 250 mg/ml.

Hydroalcoholic extracts of C. asiatica, R. communis and P. amarus at 125 µg/ml and 250 µg/ml show the same readings as that of the Liv-52 used as an standard at the concentration of 125 µg/ml and 250 µg/ml when compared foe ALAT, ASAT and ALP, which signifies the hepatoprotective activity of three extracts. The other two extracts,

E. alba show the same readings for ALAT at 125 µg/ml concentration while T. cordifolia shows the same readings for ASAT at 250 µg/ml concentration which signifies the small hepatoprotective activity. They have showed the better activity when compare to toxicant and they were showing similar activity when compared to control.

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Received on 03.01.2017 Modified on 18.02.2017

Res. J. Pharm. Dosage Form. & Tech. 2017; 9(2): 71-82.

DOI: 10.5958/0975-4377.2017.00014.3

Sr.No.	Type of Chemicals	Active principles	Part in which present
1.	Alkaloids	Berberine ^{18-22, 23} , Palmatine^18-22 , holine ^18-22Tinosporine ^18-22 , Palmatine ^23.	Stem Root
2.	Glycosides	18-norclerodane glycoside²⁴, Furanoid diterpene glucoside^25,26, Tinocordiside^27-28,Tinocordifolioside^29-30, Syringin^31,32.	Stem
3.	Diterpenoid lactones	Furanolactone ³³ ,Tinosporon ³⁴ ,Columbin ³⁵	Whole plant
4.	Steroids	b - Sitosterol,³⁶ g - Sitosterol	Aerial plant stem
5.	Sesquiterpenoid	Tinocordifolin³⁷	Stem
6.	Aliphatic compound	Octacosanol³⁸ , Heptacosanol³⁸	Whole part
7.	Miscellaneous compounds	Tinosponidine ²¹, Cordifol²¹ Cordifelone²¹,Jatrorrhizine³⁹	Root whole plant

Particulars	No. of peaks	Rf value	Solvent system
Hydroalcoholic extract of Tinospora cordifolia (10 mg/ml)	4	0.17, 0.38, 0.54, 0.68	n-propanol : formic acid : water ( 90 : 01 : 09 )
Berberine (1 mg/ml)	2	0.25, 0.37	n-propanol : formic acid : water ( 90 : 01 : 09 )
Hydroalcoholic extract of Centella asiatica(10 mg/ml)	3	0.26, 0.53, 0.74	acetonitrile : methanol 1 : 1
Asiaticoside (1 mg/ml)	1	0.90	acetonitrile : methanol 1 : 1
Hydroalcoholic extract of Phyllanthus amarus(10 mg/ml)	9	0.12, 0.18, 0.27, 0.41, 0.50, 0.55, 0.68, 0.79, 0.89	acetonitrile : methanol 1 : 1
Phyllanthin(1 mg/ml)	1	0.67	acetonitrile : methanol 1 : 1

Estimation of total phenolic content:41

Procedure:

Estimation of total flavonol content:42

Estimation of total phenolic content:⁴¹

Estimation of total flavonol content:⁴²