GENERAL INVESTIGATIONS ON THE HEPATOPROTECTIVE POTENTIAL OF PHYLLANTHUS NIRURI

GENERAL INVESTIGATIONS ON THE HEPATOPROTECTIVE POTENTIAL OF PHYLLANTHUS NIRURI

Kavitha G. Singh *, N. Nandhini and Nasmah Maqsood

Mount Carmel College (Autonomous), 58, Palace Road, Ashok Layout, Vasanth Nagar, Bengaluru, Karnataka, India.

ABSTRACT: Phyllanthus niruri commonly known as Keezhanelli is traditionally used as a medicinal plant in many parts of the world. In this study, phytochemical screening was the hepatoprotective potential of the plant investigated. Two plant forms, namely, dry powder and fresh leaves were used. Aqueous and methanolic extracts were prepared from both forms, and the experiments were carried out. The hepatoprotective potential of the Phyllanthus niruri extracts was evaluated by checking the enzyme activity in CCl₄ toxicity-induced chicken liver. The liver was subjected to different extracts and the SGPT, LDH levels were calculated. The methanolic extracts displayed higher SGPT levels in the liver after CCl₄ treatment (~121 IU/L) while the dry powder aqueous extracts were the lowest (79.5 IU/L). The LDH levels were the highest for fresh leaves methanolic extracts (323.23 IU/L) while the dry powder aqueous extracts (303.01 IU/L) had the lowest level among all extracts. Thus the fresh leaves methanolic extract was proven to be the most efficient out of all of the Phyllanthus niruri extracts. All of the extracts displayed hepatoprotective activity. It can be good herbal medicine and is consumed in all forms by people.

Keywords: Phyllanthus niruri leaves, Methanolic and aqueous extract, LDH, Hepatoprotective assays

INTRODUCTION: Phyllanthu sniruri, commonly known as ‘Keezhanelli’ in Tamil Nadu and ‘Jaundice plant’, is a tropical plant that averages to around 50-70 cm in height. It has a lean appearance and bears several fruits at a time. Phyllanthus niruri Linnaus., is a small herb spread throughout the tropical and subtropical countries of the world. It is an annual plant and found mostly in the coastal areas in India. It belongs to the Euphorbiaceae family and is widely known for having medicinal properties, used across the world ¹. Phyllanthus is a large genus, consisting of more than 700 species, can be classified into 11 subgenuses.

The most popular 24 species belong to subgenus Kirganelia, Cicca and Phyllanthus and are traditionally used by different nationalities ². It has been used for a wide range of diseases in South and south‐east Asian traditional medicine, including but not limited to jaundice, diarrhoea, dyspepsia, genitourinary infections, and renal stones ³. Also found in South America, China, and South Africa ⁴. The Ayurvedic System of medicine suggests that the whole plant of Phyllanthus niruri can be used for medicinal purposes.

It has been accepted as acrid, cooling, alexipharmic. Ayurveda recommends its use for bronchitis, leprosy, anaemia, urinary discharge, asthma, etc. Local people of Chhattisgarh and Jharkhand use it for the treatment of skin diseases, indigestion, cough, ulcers etc. ⁴. The commonly used forms are either the fresh leaves that are taken from the plant while it is still at a bright green stage or the dry powdered form.

The leaves at the mature state is plucked and dehydrated, crushing them into a fine powdered texture and sold. Extracts of this herb have been proven to have therapeutic effects in many clinical studies. Hot water extract of the entire plant is administered orally to reduce fevers and as a laxative ⁵. Infusion of the green root is taken orally to treat heavy menstrual periods ⁶. Fresh plant juice is taken orally for genitourinary disorders; the fruit is used externally for tubercular ulcers, scabies, and ringworm ⁷. The dried plant's hot water extract is administered orally for diabetes ⁸ for asthma in ayurvedic medicine ⁹. Fresh leaf juice or fresh root juice are taken orally for venereal diseases. Decoction of the dried entire plant is administered orally to treat venereal diseases ¹⁰. Phyllanthus niruri exhibited many valuable properties. One among them was the anti-bacterial activity. Different extracts were prepared with the leaves, whole plant, stem, and root and then assayed with different types of bacteria to check the efficiency of the extracts. The bioactive compounds present in them proved that they had anti-bacterial properties. The whole plant extracts were the most active against the growth of the bacteria ¹¹.

Jaundice: Used by rural and tribal people ¹² the whole plant is consumed to treat jaundice.

Urinary Tract Stones: Typically used for stone risk, proved by clinical trials ¹³ where the stone size reduced, and uric acid levels decreased. Urinary calculi were eliminated.

Antiviral Activities: Studies conducted against Herpes Simplex Virus demonstrated strong antiviral activities through plaque reduction assays, and real-time polymerase chain reaction. It can also be used for the treatment of hepatitis B and HIV.

Ulcers: P. niruri has demonstrated anti-inflammatory and gastric anti-ulcer activity, particularly by the consumption of its leaves. It could reduce the size of the ulcers in rats ¹⁴.

Diabetes: In lowering the blood sugar levels and preventing the spikes in sugar level after intake of high carbohydrate-rich food. Antidiabetic property of methanolic extracts of the plant was found in a study conducted on rats. The blood sugar levels were significantly reduced when the extracts were supplied in dosage forms. They could also suppress the rise in the glucose levels after consuming a heavy glucose meal in normal rats. The rats also had lower hemoglobin levels (P<0.05) ¹⁵.

Antispasmodic Activity: Alkaloids extracted from plants of the genus Phyllanthus present an antispasmodic activity leading to smooth muscle relaxation, mostly evidenced in the urinary tract, which would facilitate the elimination of urinary calculi. These data strongly suggest that Phyllanthus niruri may be a potential source of many substances with antilithiasic properties ¹⁶.

Active Ingredients of P. niruri:

Phytochemical: Rutin, a flavonol glycoside, strengthens capillaries and so can help people suffering from arteriosclerosis or high blood pressure ¹⁷. Geranin, a tannin, possessed the ability to lower systemic blood pressure by reducing noradrenaline release in spontaneously hypertensive rats ¹⁸. Alkaloids like Nirurine, phyllanthine, and phyllochrysine were also isolated from P. niruri ^{19, 20}. A Saponin, Diosgenin is a steroid glycoside that exists widely in the plant kingdom, and plants containing this agent are extensively used in Traditional Chinese Medicine ²¹. Diosgenin demonstrated anti-fungal and cardiovascular activity ²².

Two new lignans were reported recently, namely cubebin dimethyl ether and urinatetralin, from the cell suspension cultures of P. niruri. This was the first report on cell suspension cultures of P. niruri successfully produced lignans ²³. Urinatetralin was isolated from P. niruri ²⁴. Flavonoids are used to prevent and cure diseases associated with free radicals. They are found to have a defensive effect against bacterial and viral infections and diseases such as cardiovascular ailments, malignancies, and other age-related disorder’s ²⁵.

Limonene is a monoterpenoid that has many medical and pharmaceutical applications ²⁶. The anti-carcinogenic actions of limonene in liver tumor models have been reported ²⁷. Research in Japan and India in the 1980's has demonstrated the liver-healing properties of Phyllanthus niruri. The primary compounds responsible are phyllanthin, hypophyllanthin, and triacontanal. Glycosides found in Phyllanthus niruri demonstrated Aldose reductase (AR) inhibitory activity in studies conducted by a Japanese research group in 1988 and 1989 ²⁸.

Antioxidant Properties: P. niruri extracts can break the oxidation chain reaction and inhibit peroxides' formation. The free radical scavenging activity of the plant is due to the presence of various phytochemicals like coumarins, alkaloids, coumarins which are proven to have antioxidant activities ²⁹. P. niruri can reduce oxidative stress in various organs like the kidney by preventing a decrease in the level of existing antioxidant enzymes. It is therefore beneficial in treating kidney diseases due to diabetes ³⁰.

MATERIALS AND METHODS:

Materials: Sodium hydroxide, Chloroform, Glacial acetic acid, DNPH (2,4-Diphenyl hydrazine), Sodium pyruvate,, Distilled water, Concentrated Hydrochloric acid, Sodium chloride, KRHB buffer, Carbon tetrachloride (CCl₄), Methanol, Alanine, α-ketoglutarate, Phosphate buffer, Lithium lactate, NAD, Glycine buffer. The experiment was conducted in 2021 at Mount Carmel College, Bengaluru.

Preparation of Extracts:

Dried Leaf Extract: Dried powder of Phyllanthus niruri was obtained by drying the leaves and grinding them. The ground powder was used for aqueous and Methanol extraction. 10% dried leaf extract was prepared using distilled water. 80% methanol with distilled water was used for extraction. 10% methanolic extract of dried leaf powder was prepared with 80% methanol ³¹.

Fresh Leaf Extract: Fresh leaves of Phyllanthus niruri were handpicked and cleaned. Then these leaves were ground with distilled water and 80% methanol. 10% fresh leaf extract was prepared using distilled water. 80% methanol with distilled water was used for extraction. 10% methanolic extract of fresh leaf was prepared with 80% methanol ³².

Determination of Hepatoprotective Activity:

Liver Tissue Collection: Fresh Chicken (Gallus gallusdomesticus) liver tissue was bought. A sample of liver tissue was excised and transferred immediately to ice-cold saline and transported to the laboratory on the day of the experiment in an ice bath.

Preparation of Medium: The KRHB (Krebs Ringers – 4 - (2 - hydroxyethyl) – 1 – piper –azine-ethanesulfonic acid (HEPES) ) buffer includes HEPES (2.5mM), NaCl (118mM), KCl (2.85mM), CaCl₂ (2.5mM), KH₂PO4 (1.5mM), MgSO₄ (1.18mM) and Glucose (4.0mM). The pH was adjusted to 7.4 by 1N NaOH.

Preparation of CCl₄ Solution: 9.74ml of CCl₄ was mixed with 10.26ml of distilled water to prepare 20ml of 5M solution of CCl_4. A definite volume from the stock solution was used in the reaction mixture to induce toxicity and to study its effect on liver tissue.

Experimental Design for Hepatoprotective Activity: Hepatoprotective activity of the plant extract was evaluated using the liver tissue obtained from the shop. The fresh liver was thinly sliced (500mg), washed with KRBH buffer, was transferred into different vials containing KRBH buffer and the following reaction mixtures:

1. Blank- KRBH buffer only.
2. Control- CCl₄ exposed group.
3. Dried leaf Aqueous extract and CCl₄ exposed group.
4. Dried leaf Methanol extract and CCl₄ exposed group.
5. Fresh leaf aqueous extract and CCl₄ exposed group.
6. Fresh leaf Methanol extract and CCl₄ exposed group.

All the above-mentioned cultures were incubated at room temperature for 60 minutes and then homogenized. The homogenate was then centrifuged at 4000 rpm for 15 min at 4 ^oC. The supernatants of the tissues post centrifugation were assayed for Serum Glutamate Pyruvate Transaminase (SGPT) and Lactate Dehydrogenase (LDH).

Liver Enzyme Assays:

SGPT Assay: SGPT, also called ALT (Alanine Transaminase) on incubation with Phosphate buffered substrate (0.1M, pH 7.4) containing L-Alanine and α-Ketoglutarate forms pyruvate and glutamate. The pyruvate thus formed is coupled with DNPH reagent to give hydrazine, a brown-coloured complex whose absorbance was read at 540nm. The enzyme assay was carried out by adding 1ml of buffered substrate into different test tubes which was pre-incubated at 37^oC for 15 minutes. 0.2 ml of respective supernatant was added to the respective tubes and incubated at 37^oC for 15 min.

Then 1ml of DNPH reagent was added to arrest the enzyme activity. The tubes were allowed to stand at 37^oC for 15 min. After cooling the tubes, 5ml of 0.4N NaOH was added. In the control tubes, the respective supernatants were added only after NaOH.

Similarly, the experiment was performed with standard Sodium pyruvate solution (200µg/ml) by pipetting 0.1ml to 0.5ml of the standard in different test tubes, and the final volume in each tube was made up to 1.2ml using Phosphate buffer. 1ml of DNPH reagent was added to the tubes and incubated at 37^oC for 15 min, followed by 5ml of 0.4N NaOH after cooling. The absorbance was measured at 540nm ³³.

LDH Assay: King’s method was used to determine the amount of enzyme released from the liver tissue into the medium due to toxicity induced by Carbon tetrachloride. LDH on incubation with Glycine buffered substrate (0.1M, pH10) containing Lithium lactate forms pyruvate, which reacts with DNPH reagent to give reddish complex in alkaline medium.

The absorbance was read at 540nm. 1.0ml of the buffered substrate, 0.2ml of NAD solution and 0.1ml of the respective supernatants were taken in respective tubes.

The reaction mixture was incubated at 37^oC for 15 min. Activity in each of the tubes was arrested by adding 1ml of 2,4-DNPH reagent, and the tubes were again incubated at 37^oC for 15 min. After incubation, the tubes were cooled and 7ml of 0.4N NaOH was added to all the tubes. A control having 1ml buffered substrate, 0.1ml of NAD solution, 1ml of DNPH reagent, and 7ml of 0.4N NaOH was used, after which 0.1ml of respective supernatants was added and absorbance was absorbed read. Similarly, the test was performed with standard sodium pyruvate solution (80µg/ml) by pipetting 0.05-0.25 ml of the standard in different test tubes, the final volume in each test tube was made up-to1.3ml using distilled water. 1ml of 2,4-DNPH reagent was added and incubated at 37^oC for 15 minutes, followed by the addition of 7ml of 0.4 N NaOH. The absorbance was read at 540nm ³³.

RESULTS AND DISCUSSIONS

SGPT Activity: The SGPT activity of chicken liver after CCl₄ induced toxicity and treatment with various P. niruri extracts are depicted in Table 1 as well as Fig. 1. The SGPT activity was compared with that of the chicken liver treated only with the buffer, serving as blank and chicken liver suspended in buffer and induction of CCl₄ toxicity, without adding plant extract and serving as the control.

The different preparations of the P. niruri extracts were the aqueous and methanolic extracts of the dry powder and the fresh leaves. It was observed that chicken liver which had no addition of any plant extracts and had CCl₄ toxicity induced, had the lowest SGPT activity. In contrast, the chicken liver with no toxicities induced had the highest SGPT activity.

From the graph, it was inferred that the fresh leaves methanolic extract and the dry powder methanolic extract could significantly reduce the CCl4 toxicity and display a greater enzyme activity than the other aqueous extracts. The aqueous extracts showed a lower enzyme activity, with the dry powder aqueous extracts displaying the least enzyme activity after adding plant extracts.

TABLE 1: SERUM GLUTAMIC PYRUVIC TRANSAMINASE ACTIVITY IN CHICKEN LIVER

FIG. 1: GRAPHICAL REPRESENTATION OF SGPT ACTIVITY OF CHICKEN LIVER AFTER CCL₄ INDUCED TOXICITY AND TREATMENT WITH VARIOUS P. NIRURI EXTRACTS

Lactate Dehydrogenase Assay: The LDH activity of chicken liver after CCl₄ induced toxicity and treatment with various P. niruri extracts are depicted in Table 2 and Fig. 2.

The LDH activity was compared with that of the chicken liver treated only with the buffer, serving as blank and chicken liver suspended in buffer and induction of CCl₄ toxicity, without adding plant extract, and serving as the control. The different preparations of the P. niruri extracts were the aqueous and methanolic extracts of the dry powder and the fresh leaves.

TABLE 2: LACTATE DEHYDROGENASE ACTIVITY IN CHICKEN LIVER

It was observed that chicken liver which had no addition of any plant extracts and had CCl₄ toxicity induced, had the lowest LDH activity. In contrast, the chicken liver with no toxicities induced had the highest LDH activity.

From the graph, it was inferred that the fresh leaves methanol extract and the dry powder methanol extract could significantly reduce the CCl₄ toxicity and display a greater enzyme activity than the other aqueous extracts. The aqueous extracts showed a lower enzyme activity, with the dry powder aqueous extracts displaying the least enzyme activity after adding plant extracts.

FIG. 2: GRAPHICAL REPRESENTATION OF LDH ACTIVITY OF CHICKEN LIVER AFTER CCL₄ INDUCED TOXICITY AND TREATMENT WITH VARIOUS P. NIRURI EXTRACTS

CONCLUSION: Nil

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: Nil

REFERENCES:

1. Narendra K, Swathi J, Sowjanya KM and Krishna Satya A: Phyllanthus niruri a review on its ethno botanical, phytochemical and pharmacological profile. Journal of Pharmacy Research 2012; 5(9): 4681-91.
2. Unander DW, Webster GL and Blumberg BS: Usage and bioassays in Phyllanthus (Euphorbiaceae). IV. Clustering of antiviral uses and other effects. Journal of Ethnopharmacology 1995; 45(1): 1-18.
3. Lee NYS, Khoo WKS and Adnan MA: The Pharmacological Potential of niruri 68(8): 953-69.
4. Paithankar VV, Raut KS, Charde RM and Vyas JV: Phyllanthus niruri a magic herb. Research in Pharmacy 2011; 1(4): 1-9.
5. Halberstein RA and Saunders AB: Traditional Medicinal Practices and Medicinal plant usage on a Bahamian Island. Cul Mad Psychiat 1978; 2: 177-203.
6. Singh YN: Traditional medicine in Fiji. Some herbal folk cures used by Fiji Indians. J Ethnopharmacol 1986; 15(1): 57 -88.
7. Chauhan JS, Sultan M and Srivastava SK: Two new Glycoflavones from the roots of Phyllanthus niruri. Planta Med 1977; 32: 217- 22.
8. Jain SR and Sharma SN: Hypoglycaemic drugs of Indian Indigenous Origin. Planta Med 1967; 15(4): 439-442.
9. Sircar NN: Pharmacotherapeutics of dasemani drugs. Ancient Sci Life 1984; 3(3): 132-35.
10. Holdsworth D and Wamoi B: Medicinal plants of the Admiralty Islands, Papau New Guinea. Part 1. Int J Crude Drug Res 1982; 20(4): 169-181.
11. Adebayo OL and Edwin B: Solvent Extracts of different parts of Ghanian Phyllanthus niruri show potent growth inhibition against six human pathogenic bacteria in-vitro. International Journal of Pharmacy and Pharmaceutical Sciences 2014; 6(7): 272-77.
12. Janghel V: Plants used for the treatment of icterus (Jaundice) in Central India: A review Annals of Hepatology 2019; 18(5): 658-72.
13. Pucci NC and Marchini GS: Effect of Phyllanthus niruri on metabolic parameters of patients with kidney stone: a perspective for disease prevention International Brazilian Journal of Urology 2018; 44(4): 758-64.
14. Mostafa R(2018) Ahmed S: Evaluation of anti-inflammatory and gastric anti-ulcer activity of Phyllanthus niruri (Euphorbiaceae) leaves in experimental rats. BMC Compl and Alternative Medicine 2017; 17: 267.
15. Okoli CO, Ibiam AF, Ezike A: Evaluation of antidiabetic potentials of Phyllanthus niruri in alloxan diabetic rats, African Journal of Biotechnology 2010; 9(2): 248-59.
16. Calixto JB, Santos AR, Cechinel Filho V and Yunes RA: A review of the plants of the genus Phyllanthus: their chemistry, pharmacology and therapeutic potential. Med Res Rev 1998; 18: 225-58.
17. Becker CG, Hajjar DP and Hefton JM: Tobacco constituents are mitogenic for arterial smooth-muscle cells. Am J Pathol 1985; 120: 1–5.
18. Cheng JT, Chang SS and Hsu FL: Antihypertensive action of geraniin in rats. J Pharm Pharmacol 1994; 46: 46–49.
19. Cuellar A and Estevez PV: A preliminary phytochemical study of Cuban plants. V. niruri. Rev Cubana Farm 1980; 14: 63–68.
20. Mulchandani NB and Hasarajani SA: 4-methoxy-norsecurinine, a new alkaloid from Phyllanthus niruri. Planta Med 1984; 50: 104–05.
21. Kurt BGT: Natural product chemistry: a mechanistic and biosynthetic approach to secondary metabolism In: Kurt, B. (ed.) The mevanolic pathway: the terpene. John Wiley & Sons Ltd, Chichester, New York, USA 1983; 208-09.
22. Hufford CD, Liu SC and Clark AM: Antifungal activity of Trillium grandiflorum J Nat Prod 1988; 51: 94–98.
23. Elfahmi, Batterman S, Koulman A, Hackl T, Bos R, Kayser O, Woerdenbag HJ and Quax WJ: Lignans from cell suspension cultures of Phyllanthus niruri an Indonesian medicinal plant. J Nat Prod 2006; 69: 55–58.
24. Chang CC, Lien YC, Liu KC and Lee SS: Lignans from Phyllanthusuri naria. Phytochemistry 2003; 63: 825–33.
25. Hannan JM, Rokeya B and Faruque O: Effect of soluble dietary fibre fraction of Trigonellafoenum-graecum on glycemic, insulinemic, lipidemic and platelet aggregation status of Type 2 diabetic model rat. J Ethnopharmacol 2003; 88: 73–7.
26. Singh B, Agarwal PK and Thakur RS: Triterpenoids from Phyllanthus niruri. Indian J Chem 1989; 28: 319–21.
27. Mills JJ, Chari RS, Boyer IJ, Gould MN and Jirtle RL: Induction of apoptosis in liver tumors by the monoterpeneperillyl alcohol. Cancer Res 1995; 55: 979-83.
28. Shimizu M: Studies on aldose reductase inhibitors from natural products. II. Active components of a Paraguayan crude drug Para-parai mi, Phyllanthus niruri. Chem Pharm Bull 1989; 37(9): 2531-32.
29. Yu J and Wang L: Antioxidant activity of citrus limonoids, flavonoids and cumarins. Journal of Agricultural and Food Chemistry 2005; 53(6): 2009-14.
30. Giribabu N, Rao PV: Aqueous extracts of Phyllanthus niruri leaves displays in-vitro antioxidant activity and prevents the elevation of oxidative stress in the kidney of Stretozotocin-Induced Diabetic Male Rats Evidence-Based Complementary ad Alternative Medicine 2014; 834815.
31. Kumar AK, Ramachandra SS and Narsu L: Pharmacognostic and phytochemical Investigations of roots of Hibiscus micranthus Research Journal of Pharmaceutical Biological and Chemical Sciences (RJPBCS 2010; 1(4): 324–37.
32. Harish R and Shivanandappa T: Antioxidant activity and hepatoprotective potential of Phyllanthus niruri. Food Chemistry 2006; 95(2): 180-85.
33. Aditibhattacharjee, Devi HM and Maity M: Evaluation of In-vitro hepatoprotective activity of Phlogacanthus jenkinski Goat Liver 2019; 22-26.
    

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    How to cite this article:

    Singh KG, Nandhini N and Maqsood N: General investigations on the hepatoprotective potential of Phyllanthus niruri. Int J Pharm Sci & Res 2022; 13(6): 2465-70.. doi: 10.13040/IJPSR.0975-8232.13(6).2465-70.

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