Hypotensive Activity of Moringa oleifera Lam (Moringaceae) Root Extracts and its Volatile Constituents

Purpose: To explore the hypotensive activity and chemical composition of Moringa oleifera Lam (Moringaceae) roots. Methods: The fresh roots of M. oleifera was cut into small pieces and successively extracted with petroleum ether (PE) and dichloromethane (DC). PE extract was further divided into MRP and MRP -1. DC extract showed a thick mass during evaporation which was separated as MRDC - IN. The mother liquor left was divided into MRDC and MRDC -1. All residues were analyzed by gas chromatography-mass spectroscopy (GC-MS) using ZB-5 column. Identification of each extract and fraction was based on comparison of their retention indices (RI), by co-injecting authentic compounds, as well as by comparing literature data available in NIST Standard Reference Database. Hypotensive activity was determined on urethane-anesthetized normotensive Sprague Dawly rats. Results: Petroleum ether (MRP) and dichloromethane (MRDC) extracts of M. oleifera roots showed 50.06 ± 3.48 and 48.16 ± 1.79 % fall in mean arterial blood pressure (MABP), respectively, at a dose of 30 mg/kg (p < 0.01 and p < 0.05, respectively) compared with control. GC-MS analysis of MRP and MRDC extracts and fractions resulted in the identification of seventy four (74) compounds. Methyl hexadecanoate ( 7 , 20.3 %) , stigmastan - 3, 5, diene ( 24 , 19.32 %), methyl 14-hydroxy-5-tetradecenoate ( 9 , 19.22 %), 1 , 11 diphenyl undecane ( 47 , 18.78 %) and cyclopentanyl hexadecane ( 39 , 14.44 %) were the major constituents among the various hydrocarbons, fatty acids, esters, alcohols, aldehydes, isothiocyanate, aromatics, steroids, terphenyl and sulphur-containing compounds. Conclusion: The findings reveal the hypotensive potential of M. oleifera roots and the presence of specific hydrocarbons, fatty acid esters, thioureides, steroids and isothiocyanates in active fractions. Further study is required to determine the suitability of the plant as an antihypertensive remedy.


INTRODUCTION
Moringa oleifera, Lam is one of the best known and widely utilized specie among the thirteen known species of the monogeneric family Moringaceae. It is native to Pakistan and India, and widely cultivated throughout the world [1,2].
In recent decades therapeutic preparations like Septilin for respiratory tract infection, Rumalaya for arthralgia and Pro-lacta of M. oleifera have been marketed [15]. This paper reports the hypotensive evaluation and GC-MS analysis of M. oleifera roots constituents detected in winter, which are markedly different from those analyzed during summer [16]. Earlier volatile constituents from leaves [17], flowers [18] and pods [19] of M. oleifera have already been reported in literature.

Plant material
Fresh roots of M. oleifera (10 kg) were collected in November 2007 from HEJ-ICCBS Garden University of Karachi, Karachi Pakistan. A voucher specimen (no. 66250 KUH) was deposited in the herbarium of Department of Botany, University of Karachi, where it was authenticated by Mr Abrar Hussain.

Extraction
Fresh roots of M. oleifera was cut into small pieces (1 -2 inch) and successively extracted with petroleum ether (PE) and dichloromethane (DC) at room temperature for three days. PE extract was divided into two layers during evaporation. Both layers were separated and evaporated to residual masses MRP (upper layer, 3.14 g) and MRP -1 (lower layer, 0.27 g). DC extract showed a thick mass settled at the bottom of round bottom flask during evaporation on rotavapour. It was separated, dried and weighed as MRDC -IN (2.25 g). Mother liquor left was further evaporated which when concentrated divided into two layers. Both layers were separated and evaporated to thick residues. Upper layer furnished MRDC (4.17 g) while lower layer gave MRDC -1 (8.89 g).

Gas chromatography mass spectrometry
For GC-MS 6890 N Agilent gas chromatograph coupled with a JMS 600 H JEOL mass spectrometer was used. The compound mixture was separated on a fused silica capillary ZB-5 column, (30m x 0.32 mm) 0.22 µm film thickness in a temperature program from 50 to 260 o C with a rate of 4 o C min -1 with 3 min hold. The injector was set at 240 o C and the flow rate of helium carrier gas was 1 ml min -1 . The EI mode JMS 600 H JEOL mass spectrometer had ionization volt 70 eV, electron emission 100 o A. Sample was injected manually in split mode. Ratio of sample in split mode was 1:50. Identification of each extract and fractions were based on comparison of their retention indices (RI) calculated according to the Kovats formula, using n-alkanes (C9 -C33) (sigma -Aldrich, Germany) as standards under the same chromatographic conditions and in some cases by co-injection with authentic compounds as well as by following the characteristic mass fragmentation patterns of known compounds. Retention Indices were also compared with literature data available in National Institute of Standards and Technology Standard Reference Database. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas.

Animals
Normotensive Sprague Dawley rats (both sex, 220 -250g) were housed in the animal house of Dr. HMI Institute of Pharmacology and Herbal Sciences in appropriate cages at 21 -23 o C. They were maintained at 12 h of an alternate light and dark cycle with free access to water and standard diet ad libitum. The maintenance and handling of laboratory animals and the experiments conducted follow the protocols based on internationally accepted standard guidelines of the Institutional Ethical Committee. The experimental procedures were performed according to international guidelines [20] and approved by the instutional ethical committee for handling laboratory animals (Ref no. HU/Dr.HMIIPHS/2013/11).

Hypotensive activity
Rats were anesthetized with urethane (1.2 gm/ kg i.p.). The trachea was exposed and cannulated with a polyethylene cannula to facilitate spontaneous respiration. Drugs were injected (0.2 -0.25 ml) through a polyethylene cannula inserted into the extrajuglar vein followed by a saline flush (0.2 ml). The arterial blood pressure was recorded from the carotid artery via an arterial cannula connected to research grade blood pressure transducer (Harvard, 60-3003) coupled with four channel Harvard oscillograph (Curvilinear, 50-9307)(UK). The temperature of the animals was maintained at 37 °C by over head heating lamp. The mean blood pressure was calculated as the sum of the diastolic blood pressure plus one-third pulse width. Changes in blood pressure were expressed as the percent of control obtained immediately before the administration of test substance. Acetylcholine (Ach) (Merck) at a dose of 1 µg/kg was used as positive control and atropine sulphate (0.1 mg/kg) (C. H. Beohringer Sohn Ingelheim Rhein, Germany) as muscarinic antagonist. MRP, MRP -1 and MRDC were soluble in 5 % Tween 80 and others in normal saline.

Statistical analysis
Changes in blood pressure were compared using Students t-test (IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY 2010). P < 0.05 was considered to be significant. Tables 1-2 Intravenous administration of extracts in anesthetized rats showed changes in systolic, diastolic and mean arterial blood pressure (MABP). Intravenous injection of positive control Ach at the dose of (1 µg/kg) showed (30.63 ± 3.5 %) fall in MABP while that of normal saline (0.9 % NaCl) was insignificant. Hypotensive evaluation of MRP showed significant fall in MABP at 3 mg/kg (41.84 ± 4.74 %, p < 0.01) and 30 mg/kg (50.06 ± 3.48 %, p < 0.01).

GC-MS (vide
Hypotensive effect was comparable at both doses however; duration of activity was increased with dose increments (60 sec and 120 sec) at 3

DISCUSSION
GC-MS analysis of extracts and fractions culminated in the detection of eighty five compounds out of which seventy four have been identified. Not unexpectedly, hydrocarbons, and fatty acid esters were found as commonly occurring constituents. Hydrocarbons range from C17 to C33 as di, mono and unsubstituted compounds. 1, 11 diphenyl undecane (47, 18.78 %) and cyclopentanyl hexadecane (39, 14.44 %) were major hydrocarbon of MRDC. Among fatty acid esters, methyl esters dominate and ranges from tetradecanoate to tetracosanoate. Methyl hexadecanoate (7, 20.3 %) in MRDC and methyl 14-hydroxy-5-tetradecenoate (9, 19.22 %) in MRP -1 were main esters. Isothiocyanates have been detected in all extracts except MRDC while halogenated derivatives were identified only in MRDC and MRDC -1.
The characteristic feature of MRP is the presence of new and unique thioureido polymers (14, 15, 17, 19, 22, 23) that make approximately 19 % of extract. They exhibited several mass fragments with characteristic difference of 74 amu indicating the possible loss of (-NH-CS-NH) + from corresponding molecular fragments/ molecular ion peak. Steroids (approximately 37

CONCLUSION
The hypotensive activity of non-polar extracts and fractions of Moringa oleifera roots and their chemical composition through GC-MS have been established. However, further studies are needed to ascertain its bioactivity, especially in a hypertensive model. This will help to establish the complementary effect of these components and their suitability as an antihypertensive remedy.