Abstract
The volatile constituents, toxicity, antinociception, and anti-inflammatory activities of the essential oil obtained from the leaf of Mucuna pruriens utilis collected from Nigeria are reported. The essential oil was analyzed comprehensively utilizing gas chromatography (GC)-flame ionization detector and GC coupled with mass spectrometry (MS) using the HP-5 column. The antinociceptive and anti-inflammatory assays were analyzed by a hot plate, formalin, and carrageenan-induced edema assays, respectively. The essential oil was obtained in a yield of 0.2% (v/w) calculated on a dry weight basis. A total of 36 compounds representing 94.8% of the oil contents were identified. The oil contained a high content of (E)-2-hexenal (19.0%), linalool (8.9%), 1-hexanol (6.6%), and trans-dehydroxylinalool oxide (5.2%). The analgesic property of the essential oil was slightly significant (P < 0.5) only at the third hour for the 400 mg/kg while other doses are less active. The rate of inhibition was moderate (24.1%-54%) during the analgesic phase of the formalin assay. The rate of inhibition at the anti-inflammatory phases of both formalin and carrageenan were significantly high (100%) and P < 0.001 for all the doses during the reaction duration. The potential proinflammatory mechanism might be due to effects on several proinflammatory mediators, including, histamine, serotonin, and bradykinin, and the ability of the essential oils to act as centrally mediated opioid analgesic. Mucuna pruriens essential oils displayed a high anti-inflammation potential and can be used as a potential centrally mediated opioid antagonist against analgesia.
Introduction
Mucuna pruriens, a member of the family Fabaceae, belongs to the genus Mucuna. It is commonly known as velvet bean, cowage, cowitch, or Lyon bean. 1 In Nigeria, it is well known as “Ewe Ina” among the Yoruba’s. Globally, this species is widely distributed in the tropical region of Asia and pantropical. It occurs in most of India and the seeds are used to maintain male libido activity. 2 Besides, the local tribes of the Benin Republic and Vietnam apply it for the pest grass, Imperata cylindrical. Mucuna pruriens displays several pharmacological properties like analgesic, anti-inflammatory, antineoplastic, antiepileptic, and antimicrobial activities. 3 -5 Several phytochemicals such as tannins, alkaloids, phenolics compounds, terpenoids, and flavonoids are present in M. pruriens. 6
Traditional use of the plant as antivenom is prevalent among the Northern tribes of Nigeria. The methanolic extract of the seed successfully neutralizes the effect of Echis carinatus and Naja sputatrix venoms in mice and rabbits by employing the immunological neutralization and protective mechanism.
7,8
Also, the M. pruriens seeds have demonstrated various anti-Parkinson’s activities due to the high presence of
Phytochemical screening of M. pruriens seed aqueous extract using the high-performance liquid chromatography shows the presence of proanthocyanidin, tannin, gallic acid, quercetin, and phytic acid. Mucuna pruriens seed extract is a source of
Materials and Methods
Collection of Leaf Sample
The leaves of M. pruriens were collected from the Imota Ikorodu Forest Reserve (6.6636oN, 3.6699oE), Lagos State Nigeria, in November 2018. The Curators’ authentication and identification is at the Herbarium Headquarters, Forestry Research Institute of Nigeria (FRIN), Ibadan, Nigeria, where a voucher number, FHI 111386, was deposited.
Preparation of Plant Sample
Prior to the hydrodistillation process, the plant samples were air-dried under laboratory shade for 2 weeks (at 25°C) to reduce the moisture contents. In addition, sediments and other unwanted materials were separated from the samples. Afterward, samples were pulverized to a coarse powder.
Hydrodistillation of the Essential Oil
In this process, 345.0 g of air-dried and pulverized leaves of M. pruriens were used. Hydrodistillation was carried out with a Clevenger-type distillation unit designed according to the specification. 22 The oil was distilled over water and collected in hexane at normal pressure at about 100°C. The distilled volatile oils were collected in a tight screwed amber bottle and weighed. The oils were kept under refrigeration (4°C) until the moment of analyses.
Chemical Analysis of the Essential Oil
GC analysis of the oils
GC analysis was accomplished with an HP-5890 Series II instrument equipped with an HP-Wax and HP-5 capillary columns (both 30 m × 0.25 mm, 0.25 µm film thickness), working with the following temperature program: 60°C for 10 minutes, rising at 5°C/minute to 220°C. The injector and detector temperatures were maintained at 250°C; carrier gas nitrogen (2 mL/minute); detector dual, flame ionization detector (FID); split ratio 1:30. The volume injected was 0.5 µL. The relative proportions of the oil constituents were percentages obtained by FID peak area normalization without the use of a response factor.
GC–MS analysis of the oils
GC-electrospray ionization-MS analysis was performed with a Varian CP-3800 gas-chromatograph equipped with an HP-5 capillary column (30 m × 0.25 mm; film thickness 0.25 µm) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions: injector and transfer line temperature 220°C and 240°C, respectively; oven temperature programmed from 60 to 240°C at 3°C/minute; carrier gas helium at a flow rate of 1 mL/minute; injection volume 0.2 µL (10% n-hexane solution); split ratio 1:30. Mass spectra were recorded at 70 eV. The acquisition mass range was m/z 30-300 at a scan rate of 1 scan/second.
Identification of the constituents was based on a comparison of the retention times with those of authentic samples, comparing their linear indices relative to a series of n-alkanes. Further identifications were also made possible by the use of a homemade library of MS built up from pure substances and components of known oils, and MS literature data as described previously. 23 Moreover, the molecular weights of all the identified substances were confirmed by GC-chemical ionization (CI)-MS, using methanol as CI ionizing gas.
Drug and Chemicals
Carrageenan of analytical grade was obtained from Sigma-Aldrich Chemical Co. (St Louis, MO, USA; Batch Number: SLBR0530V). Piroxicam (May and Baker; Batch Number: MT2056) and ibuprofen injection (Dizpharm, Nigeria Ltd., Batch Number: 180606) were purchased from Juta Pharmacy, Lagos, Nigeria.
Animal Study
Wistar rats of about 150-200 g of Wistar rats of both sexes were provided by the Biochemistry Department animal unit and accommodated within the animal facility of Lagos state University, Ojo-Lagos. Animals were assigned at random to a group of 5 consisting of 6 animals per group and kept in a metal steel cage, with unlimited supply to water and standard pellet food. They were acclimatized for 2 weeks before the commencement of the experiment.
Group 1—ibuprofen/piroxicam-treated group 100 mg/kg (standard group); group 2—control group (saline solution, 0.9%); group 3—100 mg/kg of M. pruriens essential oil (MPEO); group 4—200 mg/kg of MPEO; and group 5—400 mg/kg of MPEO. Saline (0.9% sodium chloride) was used as a vehicle for the administration of standards, extracts, and as a control for all the assays. “Normal” saline is safe. It does not exert any physiological changes to the animal. 24
An ethical clearance certificate was obtained from the Research Ethical Clearance Committee (RECC) of the University (approval no: 012/2019/LASU/BCH).
Carrageenan-Induced Paw Edema in Rats (Anti-Inflammatory Analysis)
Animals in each aforementioned groups were induced subcutaneously with 0.1 mL of 1% freshly prepared carrageenan in the right hind paw of rats treated by oral administration of vehicle normal saline (10 mL/kg; control), sodium diclofenac (10 mg/kg; positive control), and MPEO doses (100, 200 and 400 mg/kg) with normal saline as vehicle following Avoseh et al’s method with slight modification. 25,26 Paw volume of the injected rats was measured hourly on a plethysmometer (Ugo Basile, Italy mod. 7140) commencing 1 hour before (basal values) and up to 4 hours following carrageenan injection. Edema was calculated as the difference (L) between injected and control paw. The area under the curve (AUC) time versus change (Δ) in paw volume was calculated for each animal, and the edema was expressed as the mean ± standard error of the mean (SEM) of AUC. 27
Toxicity Assay
The essential oil was tested for acute toxicity study. Twenty-five Wistar rats (both sexes, 150-200 g each) divided into 5 animals into each group were used for the toxicity study. Wistar rats were administered 500, 1000, 1500, and 2000 mg/kg of the MPEO peroral route. One group received normal saline that served as a negative control. 23
Hot-Plate Test for Antinociceptive Study
The experiment was carried out according to the modified method. 25 Thirty mature Wistar rats (both sexes) were randomly divided into 5 groups of 6 rats per group. The animals were fasted for 12 hours with the provision of clean water ad libitum. Each rat was placed upon the heated metal plate (hot plate) maintained at the temperature of about 50-55°C 15 within the restraining glass cylinder. Group 1 rats received 10 mL/kg of saline solution and served as control. Group 2 rats received 10 mg/kg of piroxicam (standard control), and groups 3, 4, and 5 received 100, 200, and 400 mg/kg of M. pruriens extract with normal saline as vehicle, respectively per os (p.o.). Animal response to the heat varies, and such changes include kicking of the hindfoot and jumping about, licking of the foot, raising the foot, holding the foot tightly to its body, or shaking of the foot. The reaction time was recorded 30, 60, 90, and 120 minutes after the administration of the treatments. The maximum reaction time was fixed at 30 seconds to prevent any injury to the tissues of the paws. If the reading exceeds 30 seconds, it is taken as a maximum analgesia.
Formalin Test
This test was based on the method of Avoseh et al’s with slight modification. 28 Briefly, formalin solution (10 mg/L in normal saline solution 0.5 mL/paw) was injected into the hind paw plantar surface (intraplantar injection) of the grouped animals. The time (seconds) spent in intense licking or biting the affected paw was rated during 2 time intervals: 0-5 minutes (first phase or neurogenic pain) and 15-30 minutes (second phase or inflammatory pain). Oral treatment (p.o.), with piroxicam (10 mg/kg; positive control), vehicle normal saline (10 mL/kg), and MPEO (100, 200, 400 mg/kg), was given 60 minutes prior to formalin injection. Percentage inhibitions are evaluated using:
where T is the number of times treated mice licked/bite the injected paw and C is the number of times control mice licked/bit the treated paw.
Statistical Analysis
Unless otherwise specified, data represented the mean ± EM of the evaluated parameter and were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple for the formalin-induced assay. At the same time, 2-way ANOVA and post hoc Bonferroni tests were used for the carrageenan and the hot-plate test using GraphPad Prism (version 7.02; San Diego CA, USA, www.graphPad.com). The minimum level of significance considered was P < 0.05, P < 0.01, and P < 0.001 were considered as the statistically significant difference of the means.
Results and Discussion
The average yield of the light-yellowish essential oil was 0.2 (v/w), calculated on a dry weight basis. The chemical constituents present in oils were indicated by their percentages as well as linear retention indices on the HP-5 column. The GC/GC–MS of essential oils of M. pruriens contains a total of 36 compounds representing 94.8% of the total oil contents identified. The main classes of compounds identified in the oil were nonterpene derivatives (35.7%), oxygenated monoterpenes (33.0%), sesquiterpene hydrocarbons (1.8%), oxygenated sesquiterpenes (10.3%), oxygenated diterpenes (3.2%), and apocarotenoids (10.8%) as shown in Table 1. The main constituents of the oil were (E)-2-hexenal (19.0%), linalool (8.98%), 1-hexanol (6.6%), and trans-dehydroxylinalool oxide (5.2%).
Compounds Identified in the Essential Oils of Mucuna pruriens.
LRI, Linear Retention Indices.
aElution order on HP-5 column.
bRetention indices on HP-5 column.
dCorrect isomer not identified.
The essential oils of M. pruriens leaves are yet to be reported; however, Arasu et al, macerated the plant sample in chloroform:methanol (2:1) solvent to obtain a mixture of nonterpenoid constituents with hydroxylamine (88.0%) as the major product. 31 Supercritical carbon dioxide extraction of the seed oils of Mucuna deeringiana shows the presence of fatty acid compounds. The primary fatty acids were linoleic acid (about 40%), palmitic acid (about 20%), and oleic acid (about 16%). 32 Jhariya and Kakkar 33 analyzed bioactive components of the ethyl acetate extract of M. pruriens using GC–MS; the study reveals the presence of hexadecanoic acid, methyl ester, n-hexadecanoic acid, 9(Z),12(Z)-octadecadienoic acid, and pyrrolidine,1-(1-oxo-7,10-hexadecadienyl). A recent study on the volatile oils of the leaves and stem of M. sloanei shows the presence of α-terpineol (18.1%) and nerol (17.5%) as the major constituents of the leaves while hexadecanoic acid (25.9%) and 9(Z),12(Z)-octadecadienoic acid (16.7%) characterized the stem. 21 The essential oils constituents of M. pruriens obtained in this study, contain hydrocarbons such as n-dodecane, tridecane, pentadecane, n-heptadecane, and n-nonadecane in small amounts which has been previously reported in the essential oils of M. sloanei. The main components identified in this study, (E-2-hexenal) and linalool, are common volatile components in some plant species. 34 -37 E-2-hexenal is a common nonterpenoid component in plant leaves, and it imparts high pharmacological properties. 38,39 Phytochemical screening of M. pruriens has shown the presence of nonvolatile terpenoids such as glutathione, gallic acid, and beta-sitosterol in the leaves extract. 40 Essential oil compositions and quality in a plant genus are influenced by several factors such as climate, plant maturity, type of soil, method of isolation, and species of a plant analyzed. These factors could account for the difference in essential oils composition of M. pruriens and other species in the genus.
Toxicity Effect
The acute toxicity of the essential oils evaluated at 500, 1000, 1500, and 2000 mg/kg body weight showed no contrary effects on the behavioral responses in the tested rats following 14 days of observation. No mortality, size, or weight change observed. Therefore, the highest dose of 400 mg/kg was adopted to be administered to rats in this study were considered safe. This result is consistent with the earlier report by Nweze et al 41 who reported no mortality or toxicity when 10, 100, 1000, 1600, 2900, and 5000 mg/kg body weight of M. pruriens extract, were orally administered to Albino rats.
Antinociceptive Assay
This assay is to evaluate the analgesic ability of plant extracts. It also depicts the strength of the plant extract to act as an opioid antagonist. Opioid receptor antagonists block one or more of the opioid receptors in the central or peripheral nervous system. The three most clinically relevant opioid receptors are the µ, κ, and δ receptors. 42 In the hot-plate test, pain induced by the thermal stimulus is to evaluate the centrally mediated analgesia characteristics of opioid. The tests are objective, quantifiable, and can be administered repeatedly without causing inflammation, and assesses supraspinally organized responses to a noxious stimulus. 43 Figure 1 shows the effect of MPEO on the thermal stimuli of Wistar rats at different exposure times.
Effect of Mucuna pruriens essential oil (MPEO) on heat-induced pain. Control, standard, and MPEO represent 1 mL of saline solution, 100 mg/kg of peroxicam, and 100, 200, and 400 mg/kg of essential oils of M. pruriens, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 statistically compared with control.
The result shows that the time taken by the saline-treated Wistar rats for which it remained on the hot plate was significantly low compared with the standard (piroxicam). Whereas, when the rats were treated with MPEO, mice stayed on the hot plate significantly longer than the control rats (P < 0.5) (Figure 1). Our findings suggested that MPEO-treated rats can resist the thermal pain, although activities were nonsignificant for most doses except at the 30th minute for the 400 mg/kg. This behavior could be predicated on the ability to hinder the synthesis or liberation of some pain mediators. The presence of mono and sesquiterpenoids in the oil could be responsible for the activity observed as previously reported. 43 Synergistic interaction of the components of MPEO could be responsible for the observed change. However, abundant constituents such as (E)-2-hexenal and linalool with previously reported pharmacological properties could be accountable. 2(E)-hexenal has antimicrobial activity against Salmonella enteritidis, Escherichia coli, Listeria monocytogenes, and Aspergillus flavus and are used to extend the shelf life of fruits such as apple. 38,44,45 Linalool’s antinociceptive properties have been reported in several models of neuropathic pains. Peana et al observed that opioidergic and cholinergic systems activation were involved in the antinociceptive effect of linalool. 46 Besides, Batista et al showed that linalool successfully reduces the mechanical pain triggered by the hypersensitivity induced by neuropathic pain and also inhibits the proinflammatory cytokines. 47 This study has shown in part that MPEO is a mild centrally mediated nonsteroidal antinociceptive extract suppressing the pain mediators.
Formalin-Induced Assay
The formalin test for nociception, usually used with rats and mice, involves moderate, continuous pain generated by injured tissue. This test is a handy tool for assessing the antinociceptive properties of drugs and for elucidating the action mechanism. The responses to formalin-induced pain, such as licking and biting of the injected paw, are biphasic. The first (early) phase is caused predominantly by C-fiber activation reflecting centrally mediated pain with the release of substance P. On the other hand, the second (late) phase depend on a combination of ongoing inputs from nociceptive afferents, due to the release of excitatory amino acids, prostaglandin E2, nitric oxide (NO), and other inflammation mediators. 48 Therefore, it allows analysis of drug actions relevant to acute and toxic pain during one test. Reports have shown that drugs that act mainly centrally, such as opioids and narcotics, inhibit both phases of formalin-induced pain while peripherally acting drugs, only restrain the late phase.
In the present study, the reactions of MPEO induced Wistar rats to formalin inhibition are shown in Table 2. At the early phase (neurogenic), the essential oils show a low to moderate inhibition averaged between 31.9% and 54.6%, which is relatively small. The highest peak is by the 400 mg/kg essential oils dose with an inhibition rate of 54.6%. The reduced activities recorded are in agreement with the hot-plate model earlier reported in this study. At the latter phase (anti-inflammatory), the essential oils significantly inhibited the release of inflammation mediators at a maximal value for all the doses >95.6%. Since essential oil of M. pruriens was effective in suppression of the latter phase of the formalin test, it can be seen that the anti-inflammatory activity of M. pruriens is mediated peripherally. Essential oil components have been recorded as excellent neurogenic and anti-inflammatory inhibitors. At a dose of 10 and 50 mg/kg, methanolic extract from the seed powder of M. pruriens successfully inhibited acute inflammation induced by formalin at 6.6% and 38.8%, respectively. 49 In addition, Iauk et al showed that the ethanolic extract of M. pruriens increased the pain threshold and also reduced body temperature. 15 Essential oil components are excellent pain mediators. Successful inhibitions had been reported for the components in these essential oils; for example, linalool from Ocimum basilicum causes peripheral and central opioid activity, and E-nerolidol shows peripheral activities. 50 The action of MPEO in this report shows that it inhibits both phases at varying degrees, which supports that the MPEO behaves as a centrally mediated pain inhibitor.
The Statistical Result of the Formalin-Induced Assay of MPEO on Wistar Rats.
MPEO, Mucuna pruriens essential oil.
Anti-Inflammatory Assay
To evaluate the anti-inflammatory activity of MPEO, the carrageenan-induced model was adopted. Carrageenan is large, highly flexible molecules that form curling helical structures that are used as phlogistic agents for the release of inflammation and proinflammatory mediators such as prostaglandins, leukotrienes, histamine, cytokines, etc. It is a well-defined model for acute inflammation, and it is used in the study of the antiedematous effect of extracts due to the production of different inflammatory mediators in the Wistar rat. The model is time-dependent, characterized by the biphasic release of mediators. The initial phase involves the release of mediators such as histamine, serotonin, and bradykinin; it lasts within the first 1 hour, while 2-4 (latter period) is characterized by infiltration of leukocytes and prostaglandins biosynthesis. 51
In this study, MPEO significantly inhibits the development of edema at all stages of exposure except for the 400 mg/kg extract, as shown in Figure 2. The results show that MPEO exhibit maximum activity within the 1-3 hours of administration, indicating its ability to suppress the expression of histamine, serotonin, and bradykinin (0-1 hour post-treatment), while the latter phase is suspected to be inhibition of leukocytes and elevated prostaglandins biosynthesis(second to fourth hour post-treatment). The reduction in activity at longer hours can be a result of the high absorption capacity of the oil by the macrophages because of its high volatility and its inhibitory capabilities.
Effect of Mucuna pruriens essential oil (MPEO) on carrageenan-induced inflammation. Control, standard, and MPEO represent 1 mL of saline solution, 100 mg/kg of diclofenac injection and 100, 200, and 400 mg/kg of essential oils of M. pruriens, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 statistically compared with control.
Ethanolic extract of M. pruriens had been shown to significantly (P < 0.001) reduce carrageenan-induced paw edema in rats at the early stage of inflammation resulting in suppression of histamines and serotonins. 52 The powdered seed also showed a very high anti-inflammatory activity against carrageenan-induced paw edema for more than 3 hours from samples collected in Pakistan. 14 Uchegbu et al also reported a high percentage reduction in edema size of between 45% and 50% when treated with seed powder of M. pruriens from Nigeria. 49 This study agrees with our finding to validate the high anti-inflammatory properties of the volatile oils of M. pruriens.
The activities of essential oils against inflammatory conditions is majorly attributed to the plant’s major constituents or in synergy with other constituents. The volatile oils of Mimusops elengi flower containing E-2-hexenal as one of the major products selectively inhibited the cyclooxygenase; COX-1 and COX-2 at 66% and 27% inhibition rate, respectively. 53 In a dose-dependent manner, E-2-hexenal showed a pronounced antifungal activity against Aspergillus flavus, and such antimicrobial activity is attributed to the highly reactive electrophilic α,β-unsaturated carbonyl moiety. 38,39,54
Conclusion
Essential oils were successfully isolated from the leaves of M. pruriens via the hydrodistillation method. The essential oils act by increasing the level of thermal response in a hot plate antinociceptive model, thereby suppressing the release of some pain mediators at the macrophages by acting in the central nervous system, thus displaying an opioid antagonist character. The anti-inflammatory efficacy, analyzed by the formalin model and carrageenan-induced edema, reveals that the essential oils were more effective for the anti-inflammatory stages with an inhibition rate of about 100% that is close to that of the standard drug, piroxicam, and ibuprofen. This investigation shows that essential oils from M. pruriens leaves can be used as a drug to modulate various inflammatory mediators such as cytokines, prostaglandins, NO, histamine, and serotonin. Further studies are needed to provide and interpreted the in vitro molecular mechanism of the inflammation.
Footnotes
Acknowledgements
The authors would like to express their appreciation to the Department of Biochemistry, Lagos State University, for their technical assistance during the animal study and Prof. Guido for technical assistance in GC/MS analysis. The authors gratefully acknowledge the financial support received from the Office of Research Directorate, Vaal University of Technology, Vanderbijlpark, South Africa.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
