ANTIHYPERTENSIVE ACTIVITY GUIDED-FRACTIONATION OF NARDOSTACHYS JATAMANSIHTML Full Text
ANTIHYPERTENSIVE ACTIVITY GUIDED-FRACTIONATION OF NARDOSTACHYS JATAMANSI
B. B. Rachotimath 1, M. S. Gavimath 2 and B. Padmashali * 1
Department of Chemistry 1, School of Basic Sciences, Rani Channamma University, Belagavi - 591156, Karnataka, India.
Department of Rasashastra and Bhaishajya Kalpana 2, SBSS Krishna Ayurvedic Medical College and Hospital, Sankeshwar - 591313, Karnataka, India.
ABSTRACT: The present study aimed to examine the anti-hypertensive constituents present in Nardostachys jatamansi. Crude solvent extracts (dichloromethane; ethyl acetate; methanol, and water) of this plant rhizomes based on the difference in polarity were prepared and tested for inhibition of angiotensin-converting enzyme (ACE). Preliminary phytochemical analysis showed bioactive compounds such as alkaloids, flavonoids, carbohydrates, triterpenoid, glycosides, saponin, and tannins in different extracts. Ethyl Acetate extract has shown better results with 93% inhibition at 100 µg concentration as compared to other solvent extracts. Fractionation of ethyl acetate extract over silica gel column chromatography with hexane and ethyl acetate under gradient elution resulted in six fractions. However, on purification of the ethyl acetate extract, ACE inhibition activity diminishes with only 19.03 to 47.35% inhibition for the purified fractions. Nardostachys jatamansi can be used to treat hypertension-related disorders, and ethyl acetate extract can be further investigated to identify potent molecules that work in a synergistic combination.
Nardostachys jatamansi, Angiotensin converting enzyme (ACE), Hypertension, Extracts, Column chromatography
INTRODUCTION: Nardostachys jatamansi (family Valerianaceae) is a perennial herb found in Alpine Himalayas. It is a reputed Ayurvedic herb and have demonstrated hypotensive, hypolipidemic, antimicrobial, antiarrhythmic, hepatoprotective, etc. 1-8 Ethanol extract of the roots of N. jatamansi DC was studied for its anticonvulsant activity and neurotoxicity demonstrated a significant increase in the seizure threshold by N. jatamansi DC root extract against maximal electroshock seizure (MES) model as indicated by a decrease in the extension/flexion ratio 9.
Chemical examination of this plant revealed the presence of terpenoid ester, nardostachysin N, volatile, non-volatile constituents, sesquiterpenes coumarins, lignans, neolignans, alkaloids. Acacin, Ursolic acid, Octacosanol, Kanshone A, Nardosinonediol, Nardosinone, Aristolen-9beta-ol, Oleanolic acid, Beta-sitosterol are chemical constituents isolated from this plant 10-12. The Discovery of the angiotensin-converting enzyme (ACE) leads to the drug discovery of anti-hypertensive drugs. The ACE converts angiotensin decapeptide inactive form into active octapeptide angiotensin II in the kidneys, especially in the renin-angiotensin-aldosterone system 13. Captopril, Enalapril, Zofenopril, Ramipril, Fosinopril, Lisinopril, and SQ 29852 are the well-known ACE inhibitors 13. Generally, Captopril is used as a positive control in most of the research study owing to its free radical scavenger activity that is highly relevant as an ACE inhibitor 13.
Considering the above facts, the present study is aimed to conduct phytochemical screening and to examine the anti-hypertensive constituents present in rhizomes of Nardostachys jatamansi.
MATERIALS AND METHODS:
Plant Material: Nardostachys jatamansi rhizomes were collected in the month of June 2016 from High Altitude Plant Physiology Research Centre, Srinagar [Gharwal] Uttarkhand, India. Rhizome was authenticated by comparing with the Herbarium (No. 2016/52) maintained at Department of Botany, J.S.S College Belagavi, Karnataka, India.
Extraction: The rhizomes were coarsely powdered in a pulverize with sieve # 16 in K.L.E Ayurveda Pharmacy, Shahapur, Belagavi, Karnataka, India. 100 g of each powdered material was subjected to Soxhlet extraction by using 400 mL of different solvents viz. dichloromethane, ethyl acetate, methanol, and water individually.
The extracted solution was dried individually under vacuum using a rotary vacuum evaporator and preserved in different amber-colored bottles.
Phytochemical Screening: The preliminary phyto-chemical screening was performed to characterize the various phytochemicals present in Nardostachys jatamansi rhizomes 14. For instance, for the determination of alkaloids, Mayer’s test, Hager’s, and Dragendroff’s test were performed.
Ferric chloride test was followed to identify tannins and phenolics. For the presence of glycosides, a Legal test was performed. An alkaline test was implemented to check flavonoids. Ninhydrin and Biuret test were followed to detect proteins. Steroids were checked through Salkowaski test. To test the presence of carbohydrates, Biuretic and Fehling’s tests were employed.
Fractionation and Purification: Ethyl acetate extract was fractioned over silica gel column chromatography (60-120 mesh) under gradient elution of hexane and ethyl acetate (500 mL each) and methanol (500 mL) to provide dried fractions:
Fr.1 (10% ethyl acetate in hexane); Fr. 2 (25% ethyl acetate in hexane); Fr. 3 (50% ethyl acetate in hexane); Fr. 4 (75% ethyl acetate in hexane); Fr. 5 (100% ethyl acetate) and Fr. 6 (100% methanol).
Angiotensin-Converting Enzyme [ACE] Inhibitory Assay: Solvent extracts were subjected for anti-hypertensive activity by angiotensin-converting enzyme [ACE] inhibitory method with different concentrations of extract in comparison with a standard, captopril. ACE inhibition activity was carried out at BioGenics Labs, Hubli, Karnataka, India. ACE inhibition activity measured by a UV spectrophotometer based on the rate of formation of hippuric acid from hippuryl-L-histidyl-L-leusine (hhl) catalysed by ACE 13.
Extracts were tested at 10, 50, and 100 µg dissolved in assay buffer (10mM) HEPES buffer containing 0.3 M NaCl and 10 µM zinc sulphate containing 20 µL kidney cortex plasma membranes (ACE enzyme source) and hippuryl-L-histidyl-L-leusine (hhl) as substrate. Briefly, the tested extracts are incubated at 37 °C for 10 min. Then the substrate is added, which makes a final reaction volume of 50 µL and incubated for 45 min at 37 °C. The reaction is terminated by the addition of 1M HCl. The yellow color is developed by the addition of 100 µL of pyridine and 50 µL of benzene sulphonyl chloride. The yellow color that formed is measured at 410 nm in an ELISA plate reader (iMark, BIORAD). Extracts with an inhibitory potential block the substrate availability to the enzyme and thereby cause enzyme inhibition leading to no formation of yellow color. The inhibition is represented in the form of percentage over control. Captopril, a standard, was tested at a concentration of 10, 15, and 25 nM. The rate of ACE inhibition was calculated by the formula,
% Inhibition = Δ Control absorption – Δ Sample absorption / Δ Sample absorption
RESULTS AND DISCUSSION: Preliminary phytochemical analysis showed that the occurrence of bioactive compounds such as alkaloids, flavonoids, carbohydrates, triterpenoid, glycosides, saponin, and tannins in different extracts Table 1.
ACE inhibition at 100 µg concentration follows the order: Ethyl acetate Extract (93.23%) > Water Extract (74.23%) > Dichloromethane Extract (67.03%) > Methanol Extract (56.77%). Among all the tested extracts, ethyl acetate extract has shown better result with 93% inhibition at 100 µg concentration as compared to other solvent extracts. Captopril, a standard, was found to inhibit 85.37 % ACE at 25 nM.
Based on the ACE inhibition results, EtOAc extract was fractioned into six fractions: Fr. 1 (10% ethyl acetate in hexane); Fr. 2 (25% ethyl acetate in hexane); Fr. 3 (50% ethyl acetate in hexane); Fr. 4 (75% ethyl acetate in hexane); Fr. 5 (100% ethyl acetate) and Fr. 6 (100% methanol) Fig. 1.
TABLE 1: PHYTOCHEMICAL SCREENING OF EXTRACTS OF NARDOSTACHYS JATAMANSI RHIZOMES
|S. no.||Phytochemical Test||Dichloromethane||Ethyl Acetate||Methanol||Water|
|1||Alkaloids||+ ve||+ ve||+ ve||- ve|
|2||Tannins||+ ve||+ ve||+ ve||- ve|
|3||Saponins||+ ve||+ ve||+ ve||+ ve|
|4||Flavonoids||+ ve||+ ve||+ ve||+ ve|
|5||Carbohydrates||+ ve||+ ve||+ ve||+ ve|
|6||Glycosides||+ ve||+ ve||+ ve||+ ve|
|7||Steroids||+ ve||+ ve||+ ve||- ve|
FIG. 1: ACE INHIBITION ACTIVITY OF EXTRACTS OF NARDOSTACHYS JATAMANSI RHIZOMES. Sample 1-Dichloromethane Extract (10, 50 and 100 µg); Sample 2-Ethyl acetate Extract (10, 50 and 100 µg); Sample 3-Methanol Extract (10, 50 and 100 µg); Sample 4-Water Extract (10, 50 and 100 µg); Sample 5-Captopril (10, 15 and 25 nM) (From Left to Right)
Six fractions of ethyl acetate extract were submitted for the ACE inhibition to identify the active constituent(s). Results revealed that Fr. 5 had shown significant inhibition at 100 µg but no activity found at lower concentrations. Fr. 4 has shown a similar tendency, but inhibition at 100 µg is less than Fr. 5. Fr. 3 has shown elevation in inhibitory activity at 50 and 100 µg. The sample has shown little activity at 100 µg whereas Fr. 1, 2 have not shown any inhibition at all concentrations screened, while captopril, a standard, was found to inhibit 85.37% ACE at 25 nM Fig. 2.
It is clearly evident from the ACE inhibition activity results Fig. 1 & 2 of fractions of ethyl acetate extract of Nardostachys jatamansi rhizomes that there is a synergistic activity of constituents towards ACE inhibition. Among the tested extracts, ethyl acetate extract has shown prominent 93% inhibition at 100 µg concentration. However, on purification of the ethyl acetate extract, ACE inhibition activity diminishes with only 19.03 to 47.35% inhibition for the purified fractions (Fr.1 to Fr.6).
FIG. 2: ACE INHIBITION ACTIVITY OF FRACTIONS OF ETHYL ACETATE EXTRACT OF NARDOSTACHYS JATAMANSI RHIZOMES. Fr.1 (10, 50 and 100 µg); Fr. 2 (10, 50 and 100 µg); Fr. 3 (10, 50 and 100 µg); Fr. 4 (10, 50 and 100 µg); Fr. 5 (10, 50 and 100 µg); Fr. 6 (10, 50 and 100 µg); Captopril (10, 15 and 25 nM) (From Left to Right)
CONCLUSION: The present study was designed to examine the anti-hypertensive constituents present in Nardostachys jatamansi. Among all the tested extracts, ethyl acetate extract was found to be active against ACE inhibition. However, ACE inhibition was found to diminishes significantly on further purification of ethyl acetate extract. This suggested that potent molecules are present in ethyl acetate that works in synergistic combination and justify the use of Nardostachys jatamansifor treatment of hypertension-related disorders.
ACKNOWLEDGEMENT: Authors are thankful to Dr. U. V. Babu (Head), Dr. Neeraj K. Patel (Research Scientist), and Sharath Kumar L. M. (Research Scientist), Phytochemistry Division, The Himalaya Drug Company, Bangalore, India,
Dr. Sanjay Mishra Scientist Grade II, Prabhakar Kore Basic Science Research Centre, Belagavi for carrying out part of this research work and for fruitful discussion.
CONFLICTS OF INTEREST: The authors declare no competing financial interest exists.
- Ali S, Ansari KA, Jafry MA, Kabeer H and Diwakar G: Nardostachys jatamansi protects against liver damage induced by thioacetamide in rats. Journal of Ethnopharmacology 2000; 71: 359-63.
- Arora RB and Madan BR: Antiarrhythmics. III. Antiarrhythmic activity of Nardostachys jatamansi (an Indian indigenous drug). Indian Journal of Medical Research 1956; 44: 259-69.
- Arora RB, Sharma PL and Kapila K: Anti-arrhythmic and anticonvulsant activity of jatamansone. Indian Journal of Medical Research 1958; 46: 782-91.
- Arora RB, Singh KP, Das PK and Mistry PN: Prolonged hypotensive effect of the essential of jatamansi. Archives Internationales de Pharmacodynamie et de Therapie 1958; 113: 367-76.
- Dhiman N and Bhattacharya A: Nardostachys jatamansi (D.Don) DC.-Challenges and opportunities of harnessing the untapped medicinal plant from the Himalayas. Journal of Ethnopharmacology 2020; 246: 112211.
- Joglekar N and Barve S: Standardization, validation and elementary phytochemical screening of Valerian and Nardostachys. International Journal of Recent Scientific Research 2019; 10: 33939-42.
- Arora P, Arora V and Singh MP: Pharmacognostic standardisation of medicinally important Nardostachys Jatamansi. World Journal of Pharmacy and Pharmaceutical Sciences 2017; 6: 834-49.
- Kasar Pl, Kirte Mc and Ukey R: Effect of Jatamansi (Nardostachys Jatamansi) on blood pressure in Rajonivritti Kala (Menopause). World Journal of Pharmacy and Pharmaceutical Sciences 2018; 7: 834-41.
- Chatterjee A: The Treatise on Indian Medical Plants; National Institute of Science Communication, New Delhi, 1997.
- Tabassum K, Shafi M and Sofi G: Efficacy of Sumbul ut Teeb (Nardostachys jatamansi Linn) on vasomotor symptoms in post menopausal women-an open observational study. EC Clinical and Medical Case Reports 2.9 2019; 01-09.
- Razack S, Kandikattu HK, Venuprasad MP, Amruta N, Khanum F, Chuttani K and Mishra AK: Anxiolytic actions of Nardostachys jatamansivia GABA benzodiazepine channel complex mechanism and its biodistribution studies. Metabolic Brain Disease 2018; 33: 1533-49.
- Chatterjee A, Basak B, Saha M, Dutta U, Mukhopadhyay C and Banerji J: Structure and Stereochemistry of nardostachysin; a new terpenoid ester constituent of the rhizomes of Nardostachys jatamansi. Journal of Natural Products 2000; 63: 1531-33.
- Ahmad I, Yanuar, A, Mulia, K and Mun'im A: Review of angiotensin-converting enzyme inhibitory assay: rapid method in drug discovery of herbal plants. Pharmacognosy Reviews 2017; 11: 1-7.
- Banu KS and Cathrine L: General Techniques involved in Phytochemical Analysis. International Journal of Advanced Research in Chemical Science 2015; 2: 25-32.
How to cite this article:
Rachotimath BB, Gavimath MS and Padmashali B: Antihypertensive activity guided-fractionation of Nardostachys jatamansi. Int J Pharm Sci & Res 2021; 12(5): 2679-82. doi: 10.13040/IJPSR.0975-8232.12(5).2679-82.
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B. B. Rachotimath, M. S. Gavimath and B. Padmashali *
Department of Chemistry, School of Basic Sciences, Rani Channamma University, Belagavi, Karnataka, India.
28 April 2020
04 September 2020
11 September 2020
01 May 2021