Aphrodisiac studies of Pavetta crassicaulis Bremek. leaf, flower extract and it pure compounds using Wistar albino rats

 

Ashwathanarayana R, Raja Naika

Department of PG Studies and Research in Applied Botany, Kuvempu University, Jnanasahyadri, Shankaraghatta, Shimoga, Karnataka, India 577451.

 

ABSTRACT:

Pavetta crassicaulis Bremek.(F: Rubiaceae), an important ethno-medicinal shrub/ small tree, belonging to the family Rubiaceae , grows up to 4 meter tall the plant is endemic to India. We aimed to assess the effects of Pavetta crassicaulis Bremek. leaf and flower extract and its isolated constituent 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol and 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-  on Aphrodisiac activity. The aphrodisiac activity of Pavetta crassicaulis Bremek. leaf, flower extract and it pure compound were studied on male wistar albino rats at various dosages. The parameters observed during the study were mount frequency, mount latency, intromission frequency, intromission latency, ano-genital sniffing and genital grooming. The results revealed that, the crude extracts showed dose dependent activity in all the concentration in that, ethanolic flower extract showed excellent activity compared to ethanolic leaf extract and pure compound. The results gives the positive effect of Pavetta crassicaulis Bremek flower and leaf in the treatment of sexual disorders by the tribes of Odisha, India but the major compounds present in  2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol and 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- were not responsible for the aphrodisiac activity may be the minor compounds even though in least concentration may influence the sexual activity in tested animals.

 

INTRODUCTION:

Pavetta crassicaulis Bremek. is an important ethno-medicinal shrub or small tree belonging to the family Rubiaceae , grows up to 4 meter tall the plant. The leaves are often membranous with dark bacterial nodules, has small, white, tubular flowers, sometimes salviform or funnel-shaped with 4 spreading petal lobes. The flowers are carried on terminal corymbs or cymes, the leaves are elliptical-oblong to elliptic-lanceolate, 6-15 cm long, and pointed at both ends. The flowers are white, rather fragrant, and borne in considerable number in hairy terminal panicle which is 6-10 cm long. The sepals are very small and toothed. The flowers tube is slender and about 1.5 cm long, with obtuse petals above half the length of the tube. The fruits are black when they dry, somewhat rounded and about 6mm in   diameter ¹.

 

Pavetta crassicaulis Bremek. Scientific classification
Kingdom:	Plantae
Division:	Magnoliophyta
Class:	Magnoliopsida
Subclass:	Asteridae
Family:	Rubiaceae
Order:	Rubiales
Genus:	Pavetta
Species:	Pavetta crassicaulis Bremek.

 

In local Mizos of Mizoram, India in their folkloric medical methods Pavetta crassicaulis Bremek plant parts like bark pulverized or in decoction is used for visceral obstructions arthritis. Leaves and roots used in poultices for boils and itches, decoction of leaves used externally for hemorrhoidal pains. The root of this plant is bitter and is given in visceral problems and dropsy. The bark is used on the victims of epilepsy. Decoction and boiled leaves are used to cure hemorrhoids, urinary complaints, and anticephalagic. Fruits used as anthelmintic and flowers are eaten fried². Rural and tribal of Odisha used Pavetta crassicaulis Bremek. leaf in the treatment of skin diseases³.

 

In Kachchh, Gujarat, India, Pavetta crassicaulis Bremek roots used by the locals and folklore medicinal practitioners in the treatment of dropsy⁴.

 

In Pankaj Oudhia’s medicinal plant data base, the local tribal medicinal practitioners in the region of Jagdalpur, Chhattisgarh, India used the treatment of hypertrophy of prostate gland, sexual disorders and in the treatment of obesity as a fat burner ⁵.

 

Local and tribal peoples of Uttara Pradesh, India, Pavetta crassicaulis Bremek roots were used in the treatment of visceral problems and dropsy. The bark is used on the victims of epilepsy. Decoction and boiled leaves are used to cure hemorrhoids⁶.

 

The study conducted in Sahyadri-Konkan Corridor of Maharashtra Western Ghats revealed the abundant distribution of Pavetta crassicaulis Bremek. in Western Ghats region of Maharashtra India⁷. The wide distribution of Pavetta crassicaulis Bremek. in Kalbhairavanatha Sacred Grove,Terungan, Ambegaon Taluka, Pune, India, a part of Western Ghats⁸.

 

A Case Study conducted on Satkosia Gorge Wildlife sanctuary and Its Surroundings, Orissa, India, on the topic of “Human induced fragment formation and Vascular Plants Species Diversity”, revealed that, the shrub/ small tree Pavetta crassicaulis Bremek. was moderately distributed among the shrub species⁹ .

 

The Preliminary qualitative phytochemical analysis of Pavetta crassicaulis Bremek. methanolic crude extract revealed the presence of Alkaloids, steroids and terpenoid with the presence of moderate antimicrobial activity^10-11.

 

Despite of meager work on this plant Pavetta crassicaulis Bremek. a very important medicinal plants were unexplored for many pharmacological activities and leaf, flower parts were used in the purpose of sexual disorder treatments (Pankajoudhia.com) in Jagdalpur, Chhattisgarh, India, so, the aim of the study was to provide data on the Aphrodisiac activity of Pavetta crassicaulis Bremek.

 

MATERIALS AND METHODS:

Study area

The study area was Sringeri – evergreen and semi evergreen forest types of Western Ghats, within Karnataka state, India with altitude range 630-840 m. Study area is situated in Chikkamagaluru (13° 26' 10.5'' N, 75° 15' 54.5'' E)

 

Plant material

The plant leaf and flower of Pavetta crassicaulis Bremek. collected from Sringeri forest, Karnataka. The botanical identification of the plant was done by Prof. K G Bhat, Udupi and the voucher specimen was conserved under the reference number KU/AB/RN/AS/002.

 

Preparation of the extract

The samples were shade dried for about 30 to 45 days and mechanically powdered. Powdered material was subjected to soxhlet¹² extraction successively with petroleum ether, Chloroform and ethanol. Samples were air dried and kept in an air tight bottles.

 

The qualitative preliminary phytochemical analysis

Extracted plant samples were screened for the presence of tannins, alkaloids, saponin, glycosides, flavonoids, steroids/sterols and   phenols   using   the   standard¹³.

 

GC-MS analysis

Plant extracts were subjected to Gas Chromatography and mass spectroscopy (GC-MS) obtained spectra was analyzed. GC Model: Thermo Trace GC Ultra, MS Model: Thermo DSQ II, Ionization: Electron Impact Ionisation (EI), Chemical Ionisation (CI), Mass Range: 1 - 1074 m/z.

 

Animals

Wistar albino rats of both sex weighing between 180 and 200 g were obtained and kept at the laboratory. The animals were maintained under standard environmental conditions and had free access to standard diet and water. Plant extracts were administered orally by gavage in distilled water at different dose levels.. The study was permitted by the Institutional Animal Ethical Committee (Reg. No. SCSCP/IAEC/11/12/2016-17), India.

 

Preparation of male rats

The male rats were trained, for sexual behavior, two times a day for a period of minimum of 10 days. The male rat which did not show any sexual interest during the test period was considered as an inactive male. The sexually active male rats were selected for testing aphrodisiac activity of the extracts.

 

Preparation of female rats

Female rats were housed in separate cages with food andwater ad libitum. The female rats were brought in oestrous phase by treating them with estradiol valerate.

 

Experimental details

The sexually active male rats were chosen separately and divided into 6 groups; each group consisting of 6 animals. The animals in the divided groups received the treatment orally. Different groups of animals which received the plant extract and the control are as follows: Control (Normal saline) 2 ml/kg b.wt, positive control (sildenafil citrate) 4.5 mg/kg b.wt, methanolic leaf extract of  the experiment plant  200 and 400 mg/kg b.wt, ethanolic flower extract 200 and 400 mg/kg b.wt, Pure compound 4.5 mg/kg b.wt.The sexual behavior of the experimental rats was observed in a dim light in specially designed cages that have glasses on all the sides and measuring 50×30×30cm. The male experimental rat was first placed in the cage and then two female rats in estrous phase were introduced. An initial period of 15 minutes was considered as acclimatization period. After 15 minutes, the extract or the drug was introduced and the activity of male rat in each group was recorded individually for 60 minutes, after 30 minutes of drug administration^14-17. To determine the aphrodisiac activity of the extracts, several parameters were observed. These include measuring and observing the mount frequency, mount latency, intromission frequency, intromission latency, ano-genital sniffing and genital grooming.

 

Mount frequency

Mounting is defined as the climbing of one animal by another usually from the posterior end with the intention of introducing one organ into another. Mount may also be operationally defined as the male assuming the copulatory position but failing to achieve intromission. Mount Frequency (MF) is therefore defined as the number of mounts without intromission from the time of introduction of the female until ejaculation.

 

Intromission frequency

Intromission is the introduction of one organ or parts into another. E.g. the penis into the vagina. Intromission Frequency (IF) is therefore defined as the intromissions from the time of introduction of the femaleuntil ejaculation.

 

Mount latency

Mount latency (ML) is defined as the time interval between the introduction of the female and the first mount by themale.

 

Intromission latency

Intromission latency (IL) is the time interval from the time of introduction of the female to the first intromission by themale. This is usually characterized by pelvic thrusting, and springing dismounts.

 

Ano-genital sniffing

Ano-genital sniffing (AS) is defined as sniffing of genital part by the test male rat after the introducing the test drug. Ano-genital sniffing count is the number of sniffing of Genitals in a given interval of time were recorded.

 

Genital grooming

Genital grooming (GG) is licking of male genital part by male rat after test drug administration. So, genital grooming count is the number of grooming ofgenital in a given interval of time were recorded.

 

                                         Number of mounted

Percentage mounted= -----------------------------------X100

                                           Number of paired 

 

                                         Number of intromissions

Percentage intromitted=---------------------------------X100

                                          Number of paired 

 

                                   Number of intromissions

Intromission rate= -----------------------------------X100

                      Number mounts+ Number of intromissions

 

 

Acute toxicity studies

Rats of both sex (three females and three males, weight: 25–35 g, age: 6–8 weeks) received ethanolic extract of Pavetta crassicaulis Bremek. leaf and bark starting at 2 g/kg orally by gavage. The animals were observed for toxic symptoms continuously for the first 4 h after dosing. Finally, the number of survivors was noted after 24 h and these animals were then maintained for further 13 days with observations made daily¹⁸. The acute toxicity studies were carried out according to OECD guidelines – 425.

 

Statistical analysis

The mean value ±SEM was calculated for each parameter. Results were statistically analyzed by one way analysis of variance (ANOVA) followed by Dunnet’s t-test. Symbols represent statistical significance.*P < 0.05, ** P< 0.01, ***< 0.0001, ns - not significant, as compared to control group.

 

RESULTS:

Extract yield and Preliminary phytochemical analysis

The soxhlet extraction of Pavetta crassicaulis Bremek. leaf (750 g) with petroleum ether gives 12.32 g, with chloroform gives 19.99 g and with ethanol gives 75.35 g yield. The results of preliminary qualitative phytochemical screening of different extracts of Pavetta crassicaulis Bremek. leaf indicate the presence of Saponins, Tannins, Flavanoids, Steroids/Sterols, Glycosides and Phenols in ethanolic crude extract and in chloroform extract confirms the presence of phenols and sterols and petroleum extracts negative results for all the phytochemical tests.

 

The soxhlet extraction of Pavetta crassicaulis Bremek. flower (750 g) with petroleum ether gives 20.34 g, with chloroform gives 18.64 g and with ethanol gives 45.38 g yield.  The results of phytochemical screening of Pavetta crassicaulis Bremek flower extracts indicate the presence of Alkaloids, Saponins, Flavanoids, Steroids/Sterols, Glycosides and Phenols in ethanol crude extract, the chloroform crude extracts shows positive results for Flavonoids. But, the petroleum ether crude extract gave negative results for all these compounds so we took ethanolic extracts for further studies (Table 1)

 

GC-MS analysis of ethanolic crude extract

a.       Leaf ethanolic extract

In GC-MS analysis of medicinal Pavetta crassicaulis Bremek. ethanolic leaf extract revealed the presence 34 compounds, in that major percentage of compounds present was 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol (38.84%), Cyclo{tetra[(5-t-butyl-2-hydroxy-1,3-phenylene)methylene]} (15.72%), 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (9.34%). The compounds such as 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol (37.44%) has anti-oxidant Antioxidant and UV stabilizing properties. Cyclo{tetra[(5-t-butyl-2-hydroxy-1,3-phenylene)methylene]} (15.72%), 4H-Pyran-4-one and 2,3-dihydro-3,5-dihydroxy-6-methyl- (9.34%) has antioxidant properties.

 

b.    Flower ethanolic extract

In GC-MS analysis of Pavetta crassicaulis Bremek.  flower ethanolic extract revealed the presence 39 compounds, in that 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (13.82%), Benzaldehyde, 2-methyl-  (7.25%), Benzaldehyde, 2-hydroxy-6-methyl- (6.52%), 2-Furancarboxaldehyde, 5-(hydroxymethyl)-(6.30%) in major percentage and it has Campesterol (0.80%), Stigmasterol (0.62%), β.-Sitosterol  (3.10%) in little percentage.

 

Acute toxicity studies

The ethanolic extract of leaf and bark of plant Pavetta crassicaulis Bremek. was found to be safe up to 3000 mg/kg body wt. by oral route. After 24 h animals were found well tolerated. There was no mortality and no signs of toxicity and extract were found to be safe.

 

Column chromatography of ethanolic leaf and Flower extract

a. Leaf ethanolic extract

Elution carried out with n-hexane in 100% concentration eluted compound 1 in small quantity and with n-hexane: ethyl acetate at the ratio 80:20 gives compound 2 but its yielded is too less. Ethyl acetate was eluted mixture of many compounds and ethyl acetate: ethanol at 80:20 yielded large quantity of compound 3. The gradient of other residues was yielded mixture of compound which is confirmed by TLC therefore not subjected to further process.

 

Compound 3L was yielded more so, we took only compound 3L for further structural analysis.

 

The pure compound 3L was crystalline transparent greyish color. Further the pure compound subjected to cNMR, hNMR, IR, Mass spectral analysis and the molecular formula was C₄₀H₅₈O₃, molecular weight 586.887 g/mol, From all these details the compound name was found to be Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol (Figure 8-11).

 

b.    Flower ethanolic extract

Elution carried out with n-hexane in 100% concentration eluted nil compound and with n-hexane: ethyl acetate at the ratio 90:10 gives compound 1 but its yielded is too less. Ethyl acetate was eluted compound 2 and ethyl acetate: ethanol at 90:10 yielded large quantity of compound 3. The gradient of other residues was yielded mixture of compound which is confirmed by TLC therefore not subjected to further process.

 

Compound 3F was yielded higher quantity so, we took only compound 3F for further structural analysis.

 

The Pure compound 3F was crystalline light brown colored and shiny surface. Further it was subjected to cNMR, hNMR, IR, Mass spectral analysis and the molecular formula was found to be C₆H₈O₄ , molecular weight 144.1253 g/mol, From all these details the compound name was found to be 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- (Figure 12-15).

 

Aphrodisiac activity

The aphrodisiac activity of ethanolic leaf and flower extracts of Pavetta crassicaulis Bremek were studied on male wistar albino rats at various dosages. The parameters observed during the study were, mount frequency, mount latency,intromission frequency, intromission latency, ano-genital sniffing and genital grooming.

 

a.     Mount frequency

The results revealed that, all the crude extracts showed dose dependent activity in all the concentration. The ethanolic flower extract (8.33±0.66) and ethanolic leaf extract (7.66±0.32) showed highest mount frequency at 400 mg/kg body weight, whereas ethanolic extracts at 200 mg/kg body weight showed moderate activity. On the other hand, the pure compounds 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol (4.5 mg/ kgbody weight) and 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-(4.5 mg/ kgbody weight) showed negligible mount frequency compared to all the extract used. Therefore, the above observation confirms the positive activity of ethanolic extracts but the major pure compounds 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol and 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- didn’t be the reason for the aphrodisiac activity.

 

b.     Mount latency

The results revealed that a significant decrease in mount latency was observed in animals treated with ethanolic flower extract (208.66±0.87) and ethanolic leaf extract (230.33±0.87) at the dose of 400 mg/kg body weight. Whereas moderate decrease was observed in animals treated with ethanolic flower extract (276.33±0.87) 200 mg/kg body weight but, the pure compounds, 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl) Phenol and 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- showed nil activity, which is not responsible for the aphrodisiac activity of crude leaf and flower ethanolic activity.

 

c.     Intromission frequency

Appreciable intromission frequency was observed in ethanolic flower extract (0.66±0.01; 1.55±0.01) and ethanolic leaf extract (0.57±0.02; 1.48±0.01)at the dose of 200 and 400 mg/kg body weight. Whereas the pure compound 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol (4.5 mg/kg body weight) and 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-(4.5 mg/kg body weight)showed nil activity.

 

d.     Intromission latency

The results revealed that the ethanolic flower extract (760±0.57; 365±2.3) and ethanolic leaf extract (666.33±1.76; 412.66±1.52) at the concentration of 200 and 400 mg/kg body weight showed appreciableintromission latency as compared to control animals. On the other hand, the pure compounds2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl) Phenol (751±8.99) (4.5 mg/kg body weight) and 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- (764.33±4.37) (4.5 mg/kg body weight)showed negligible intromission latency  in comparison to rest of the group of animals.

 

e.     Ano-genital sniffing

A significant increase in number of ano-genital sniffing was observed in the animals treated with ethanolic flower extract (6.66±0.32; 7.66±0.66) at the dose of 200 and 400 mg/kg body weight and ethanolic leaf extract (7±0.57) at the dose of 400 mg/kg body weight. Whereas pure compound at 4.5 mg/kg body weight and ethanolic leaf extract at 200 mg/kg body weight showed negligible ano-genital sniffing compared to the control group.

 

f.      Genital grooming

The results revealed that moderate increase in number of genital grooming was observed in animals treated with ethanolic flower extract (4±0.57), ethanolic leaf extract (3.33±0.87) at the dose of 400mg/kg body weight. Whereas other groups showed nil genital grooming.

 

 

Table 1: Preliminary phytochemical Analysis of different leaf and Flower extract of Pavetta crassicaulis Bremek.

Negative result, +: positive result.

 

Table 2: Effect of Pavetta crassicaulis Bremek.Leaf, Flower ethanolic extract and its pure compound on sexual behavior of male rats

Group (Dose mg/kg)	Different Aphrodisiac activity (mean ± SEM)
	Number of animals	Mount frequency	Mount latency (sec.)	Intromission frequency	Intromission latency (sec.)	Ano-genital sniffing	Genital grooming
Control	6	2.33±0.32	310±0.57	0.4±0	796±2.51	3.66±0.32	2.33±0.32
Sildenafil citrate (4.5)	6	12±0.57***	100.33±0.87***	1.74±0.02***	197±1.52***	12±0.57***	4.33±0.32***
Ethanol leaf 200	6	4±0.57	286.66±1.44	0.57±0.02***	666.33±1.76**	5.33±0.32	2.66±0.32
Ethanol leaf  400	6	7.66± 0.32***	230.33±0.87***	1.48±0.01***	412.66±1.52***	7±0.57***	3.33±0.87*
Ethanol flower 200	6	4.33± 0.32*	276.33±0.87*	0.66±0.01***	760±0.57*	6.66± 0.32**	2.66±0.66
Ethanol flower  400	6	8.33± 0.66***	208.66±0.87***	1.55±0.01***	365±2.3***	7.66± 0.66**	4±0.57*
2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol (4.5)	6	3.6±0.32	303.66±3.17	0.42±0	751±8.99*	4.33±0.32	2.33±0.32
4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-	6	4±0.57	302.33±2.82	0.51±0.01	764.33±4.37*	4±0.57	2±0.32
One way  ANOVA	P value F value	< 0.0001 44.50	< 0.0001 1716	< 0.0001 875.9	< 0.0001 3553	< 0.0001 31.46	< 0.0001 8.800

Each value is the mean ± S.E.M. of five rats. *P < 0.05, **p<0.01, ***P <0.0001 vs. control, One way ANOVA followed by Dunnet’s t-test.; % 

 

Figure  4: GC-MS chromatogram of leaf ethanolic extract of Pavetta crassicaulis Bremek.

Figure  5: GC-MS chromatogram of flower ethanolic extract of  Pavetta crassicaulis Bremek.

 

 

Table 5: Presence of metabolites in GC-MS analysis of crude ethanolic extract of pavetta leaf collected from Western Ghats Karnataka.

Sl no	Chemical compound present	Average Percentage	Properties of the compound
1	2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one	0.53	Food-grade flavor ingredients19
2	2,5-Dimethyl-4-hydroxy-3(2H)-furanone	0.52	Flavor and perfume industry20
3	2-Hexanone, 3-methyl-4-methylene-	1.45	Paint and paint thinner21
4	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-	9.34	Mutagen Antimicrobial, anti-inflammatory and antioxidant capacity22-24
5	4-[4-Chlorophenyl]-N-[2-[1-methyl-2-pyrrolidinyl]ethyl]-6-[trichloromethyl]-2-pyrimidine	1.14	Unknown
6	2,3-Dihydro-Benzofuran -	1.35	Entactogen drug of the phenethylamine and amphetamine classes, cytotoxic25
7	2-Furancarboxaldehyde, 5-(hydroxymethyl)-	0.66	Food additives, Antimicrobial, Preservative, Flavoring Agents26-27
8	1,2,3-Propanetriol, 1-acetate	1.46	Unknown
9	6-Oxoheptanoic acid	1.53	6-Oxoheptanoic acid is a reagent to synthesize new penicillin containing keto acids as side chains. It is also used to study the various metabolic pathways of 4-hydroxypentanoate and Levulinate28
10	Benzaldehyde, 4-hydroxy-	0.47	flavor and fragrance agents29
11	2-Methoxy-4-vinylphenol	1.14	Flavoring agent, antibacterial activity, anti-inflammatory effect30-32
12	Phenol, 2-methoxy-4-(2-propenyl)-	0.39	flavoring agent used in the manufacture of vanillin, Anti-Infective Agents, Anti-oxidant33-34
13	2,4-Dimethyl-3-nitrobicyclo[3.2.1]octan-8-one	0.78	Oils obtained from myrrh and frankincense and parthenium weed have little percentage35-36
14	rac-2,4-Dimethyl-3-nitrobicyclo[3.2.1]octan-8-one	0.96	Unknown
15	Benzaldehyde, 2-hydroxy-6-methyl-	0.98	Pheromone of the Acarid Mite Tyrophagus perniciosus and Grain Mite Aleuroglyphus ovatus37-38
16	2(3H)-Naphthalenone, 4,4a,5,6-tetrahydro-	0.77	Unknown
17	1,5-Diazocine, octahydro-1,5-dinitro-	0.40	Unknown
18	Methyl Ester Of Bicyclo[4.3.0]Non-1(6)-En-4,7-Dione-8-Carboxylic acid	5.66	Unknown
19	Acetic acid, (2-isopropenyl cyclopentylidene)-, methyl ester	0.83	Unknown
20	Bicyclo[3.3.1]nona-3,7-diene-2,9-dione	6.25	Unknown
21	4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol	1.32	Antimicrobial, Antioxidant, Anti-inflammatory, Analgesic26
22	2-Methyl-5-(4-methylphenyl)tetrazole	0.71	Unknown
23	2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]-	1.12	Antimicrobial, anticancer, antiinflammatory, diuretic, Cytotoxic, Flavoring Agents, used in the preparation of vitamins E and K1. It is also a decomposition product of chlorophyll, used in the treatment of arthritis39-40,23
24	Hexadecanoic acid, methyl ester	0.29	Perfumes and Cosmetics41
25	Hexadecanoic acid	2.04	Perfumes, Cosmetics, Enzyme Inhibitors,  Surfactants, Flavoring Agents, Adhesives and sealant chemicals, Agricultural chemicals (non-pesticidal), Fillers, Finishing agents, Intermediates, Lubricants and lubricant additives, Surface active agents, anti- androgenic flavor, hemolytic, 5-Alpha reductase inhibitor42-43
26	Octanal, 7-methoxy-3,7-dimethyl-	0.37	Unknown
27	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-	0.28	Anti-bacterial, Anti-inflammatory, hypocholesterolemic, cancer  preventive, hepatoprotective, nematicide, insectifuge, antihistaminic, antieczemic, antiacne, 5-Alpha reductase inhibitor, anti-androgenic, anti-arthritic, anti-coronary, insectifuge 23
28	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	0.62	Wound healing activity, Hemolytic, pesticide, flavor, antioxidant44
29	9,12-Octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester	0.30	Hypocholesterolemic, Nematicide, Anti-arthritic, Hepatoprotective, Anti- androgenic,Hypocholesterolemic, Nematicide, 5-Alpha reductase inhibitor, Anti-histaminic,Anticoronary Insectifuge,Anti-eczemic,Anti-acne45
30	2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-	0.52	Bactericide, Antifungal, Cytotoxic, Antibacterial, Antioxidant, Antitumor, Cancer preventive, Immunostimulant, Chemopreventive, Lipoxygenase-inhibitor, Perfumery, Pesticide and Sunscreen46
31	2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol	38.84	Unknown
32	Cyclo{tetra[(5-t-butyl-2-hydroxy-1,3-phenylene)methylene]}	15.72	Unknown
33	Stigmast-5-en-3-ol, (3.beta.,24S)-	0.95	Antimicrobial antioxidant, Anti-inflammatory anti-arthritic, anti-asthma, diuretic47

 

 

Table 6: Presence of metabolites in GC-MS analysis of crude ethanolic extract of Pavetta crassicaulis Bremek. flower collected from Western Ghats Karnataka.

Sl no	Chemical compound present	Average Percentage	Properties of the compound
1	2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one	1.40	Food-grade flavor ingredients19
2	1-Butanamine, 2-methyl-N-(2-methylbutylidene)-	0.48	Unknown
3	N,N'-Dimethylpiperazine	0.60	Anthelmintics, antiallergenic, antibacterial, antihistamic, antiemetic and antimigraine agents, insecticides, accelerators for rubber, urethane catalysts and antioxidants48
4	2,5-Dimethyl-4-hydroxy-3(2H)-furanone	1.99	food-grade flavor ingredients20
5	Acetic acid, 1-(2-methyltetrazol-5-yl)ethenyl ester	2.15	Anti-Arrhythmia Agents, Antifungal agents, carminative, antispasmodic, flavoring Agents, Adhesives, paint additives49
6	Butanedioic acid, monomethyl ester	0.90	Beverage industry, primarily as an acidity regulator50
7	2-acetyl-2-hydroxy-.gamma.-butyrolactone	0.73	Unknown
8	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-	13.82	Mutagen Antimicrobial, anti-inflammatory and antioxidant capacity22-24
9	Benzoic acid, ammonium salt	1.62	Industrial preservative for paper wrappers, agent for reducing curing time in vulcanization of rubber, expectorant used for chronic bronchitis, analytical reagent for various elements, Urinary anti-infective51
10	1,2-Benzenediol	4.33	Flavoring Agents, pharmaceuticals and cosmetics, antioxidant, Antibacterial52-53
11	Benzofuran, 2,3-dihydro-	3.50	Toxic54
12	2-Furancarboxaldehyde, 5-(hydroxymethyl)-	6.30	Food industry as flavoring agent55
13	1,2,3-Propanetriol, diacetate	3.00	As a sweetener; in the manufacture of dynamite, cosmetics, liquid soaps, candy, liqueurs, inks, and lubricants; to keep fabrics pliable; as a component of antifreeze mixtures; as a source of nutrients for fermentation cultures in the production of antibiotics and in medicine56
14	Benzaldehyde, 4-hydroxy-	2.30	flavor and fragrance agents57
15	2-Methoxy-4-vinylphenol	2.58	Flavoring agent, antibacterial activity, anti-inflammatory effect30-31
16	2,4-Dimethyl-3-nitrobicyclo[3.2.1]octan-8-one	0.85	Oils obtained from myrrh and frankincense and parthenium weed have little percentage35-36
17	Benzaldehyde, 2-hydroxy-6-methyl-	6.52	Pheromone of the Acarid Mite Tyrophagus perniciosus and Grain Mite Aleuroglyphus ovatus37-38
18	2(3H)-Naphthalenone, 4,4a,5,6-tetrahydro-	0.62	Unknown
19	Methyl Ester Of Bicyclo[4.3.0]Non-1(6)-En-4,7-Dione-8-Carboxylic Acid	4.77	Unknown
20	Tricyclo[7.1.0.0[1,3]]decane-2-carbaldehyde	0.88	Unknown
21	Benzaldehyde, 2-methyl-	7.25	Perfumes, Flavoring Agents57
22	2,6-Dimethyl-4-hydroxybenzaldehyde	2.73	Causes skin irritation, serious eye irritation, respiratory irritation, Herbicides, Neuropathic pain58
23	4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol	2.47	Antimicrobial, Antioxidant, Anti-inflammatory, Analgesic26
24	Neophytadiene	0.62	As additive for liquid cigarette can improve aroma and evaporation rate60
25	Hexadecanoic acid, methyl ester	1.11	Antioxidant, Hypocholesterolemic, Nematicide, Pesticide, Anti-androgenic flavor, Hemolytic,5-Alpha reductase inhibitor, Surface active agents, Laundry and Dishwashing Products46
26	N-Hexadecanoic Acid	3.11	Anti-Inflammatory, psychotropic drug, Anti-oxidant, Hypocholesterolemic, Nematicide, Pesticide, Lubricant, Anti-androgenic, Flavor, Hemolytic 5-Alpha reductase inhibitor60
27	9,12-Octadecadienoic acid, methyl ester	1.29	Anti-inflammatory, Nematicide,  Insectifuge, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic,  Anti-acne, Anti-arthritic, Anti-eczemic, 5-Alpha reductase inhibitor,  Anti-androgenic, Anti-coronary 60-61
28	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-	2.38	Anti-inflammatory, Insectifuge Hypocholesterolemic, Cancer preventive, Nematicide, Hepatoprotective, Insectifuge, Anti-histaminic, Anti-eczemic, Anti-acne, 5-Alpha reductase inhibitor, Anti-androgenic, Anti-arthritic, Anti-coronary 60
29	Alpha.-D-Glucopyranose, 4-O-.beta.-D-galactopyranosyl-	3.95	indicator carbohydrate for intestinal permeability in Crohn's disease and malabsorption syndrome61
30	2,2-Dimethyl-3-[3-methyl-5-(phenylthio)pent-3-enyl]oxirane	1.06	Unknown
31	Benzoyl .beta.-d-glucoside	2.13	Unknown
32	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	2.57	Wound healing activity, Hemolytic, pesticide, flavor, antioxidant 44
33	9,12-Octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester	2.75	Component of cigarette smoke, Hypocholesterolemic, Nematicide, Anti-arthritic,Hepatoprotective, Anti-androgenic, Hypocholesterolemic  5-Alpha reductase inhibitor, Antihistaminic, Anti-coronary, Insectifuge, Anti-eczemic,Anti-acne46
34	cis,cis,cis-7,10,13-Hexadecatrienal	1.34	Unknown
35	2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-	0.56	Bactericide, Antifungal, Cytotoxic, Antibacterial, Antioxidant, Antitumor, Cancer preventive, Immunostimulant, Chemopreventive, Lipoxygenase-inhibitor, Perfumery, Pesticide and Sunscreen46
36	Hexatriacontane	0.80	drug delivery mediums for topical and oral pharmaceuticals, organic application mediums for cosmetics, cleaning materials for art conservation, as delivery mediums and/or nutrients in nutraceuticals (vitamins and supplements), particles in personal care products (shampoo, conditioner, soap, toothpaste, etc, an crystalline fat alternative in food processing 63
37	Campesterol	0.80	Campesterol is also sometimes used to treat some specific prostate conditions, used in nutrient medicines like nutrients, body building supplements, food additive64
38	Stigmasterol	0.62	Antimicrobial, Anti-cancer, Anti-arthritic, Antiasthma Diuretic, Anti-inflammatory 46
39	β.-Sitosterol	3.10	Anabolic steroids in sports, heart disease and high cholesterol. It is also used for boosting the immune system and for preventing colon cancer, as well as for gallstones, the common cold and flu (influenza), HIV/AIDS, rheumatoid arthritis, tuberculosis, psoriasis, allergies, cervical cancer, fibromyalgia, systemic lupus erythematosus (SLE), asthma, hair loss, bronchitis, migraine headache, and chronic fatigue syndrome, anti-inflammatory, anti-cancer, antidiabetic, Antipyretic and antioxidant65-70

 

 

 

 

 

 

 

 

 

DISCUSSION:

The ethanolic extract of leaf and flower of plant Pavetta crassicaulis Bremek was found to be safe up to 3000 mg/kg hence one tenth of its dose is consider to be the safest dose for administration.

 

GC-MS analysis of Pavetta crassicaulis Bremek. ethanolic leaf extract revealed the presence 33 compounds, in that major percentage of compound was 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl)Phenol (38.84%) major percentage compound followed by Cyclo{tetra[(5-t-butyl-2-hydroxy-1,3-phenylene)methylene]}(15.72%) not reported for its pharmacological properties. The next successive compound 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (9.34%) was reported for many pharmacological properties including antioxidant properties^22-24. All the other phytocompounds were present in a meager percentage have reported for many medical as well as pharmacological properties (Table 5).

GC-MS analysis of Pavetta crassicaulis Bremek. ethanolic flower extract revealed the presence of 39 compounds. Among the all the phytochemicals present in the GC-MS analysis, 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (13.82%), Benzaldehyde, 2-methyl-  (7.25%), Benzaldehyde, 2-methyl-6 hydroxy  (6.52%), 2-Furancarboxaldehyde, 5-(hydroxymethyl)- (6.3%), were the major compounds. The major compound 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-was previously report for its anti-oxidant and other pharmacological properties (Hiramoto et al., 1997; Kumar et al., 2010; Yu et al., 2013), Benzaldehyde, 2-methyl-, Benzaldehyde, 2-methyl- 6 hydroxy, 2-Furancarboxaldehyde, 5-(hydroxymethyl)- were commonly used in food and perfume industry as a flavoring agent^37-38,55. The rest of the GC-MS compounds present in the flower ethanolic extracts were present in meager quantity has many pharmacological properties were reported (Table 6).

 

The results of this experiment revealed that Pavetta crassicaulis Bremek. plant parts definitely possesses appreciable aphrodisiac activity in all type of experimental models the major compound extracted from the leaf methanolic extract didn’t showed any aphrodisiac activity which means the major compound is not responsible for the aphrodisiac activity which is treatments used by the tribal people of Odisha⁵, but the crude extracts showed appreciable activity showed that other than the major compound may responsible for its aphrodisiac activity. Comparably flower ethanolic crude extract showed significant aphrodisiac activity than the leaf ethanolic crude extract.

 

 

 

 

Form GC-MS analysis it was revealed that the reason for the appreciable aphrodisiac activity of flower ethanolic extract may be due to the presence of Campesterol (0.80%), Stigmasterol (0.62%), β.-Sitosterol (3.10%) in little percentage.Many research papers reveled that compound like β-Sitosterol⁷¹ and stigmasterol⁷² in little concentration showed aphrodisiac activity in rats, may be the reason for the positive activity in flower ethanolic crude extract but leaf also showing the positive activity so, the active compound which influence the activity should be explored.

 

By one way ANOVA with dunnett’s comparison test revealed that both leaf and flower ethanolic extracts were significant in all the tested experiments but the pure compounds were not significant compared to the standard used.

 

Figure 8: 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl) Phenol - ¹H NMR spectrum

 

 

 

 

Figure 9: 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl) Phenol - ¹³C NMR spectrum

 

Figure 10: 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl) Phenol – IR spectrum

 

 

 

 

Figure 11: 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl) Phenol – Mass spectrum

 

 

 

 

Figure 12: 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- ¹HNMR spectrum

 

Figure 13: 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- ¹³C NMR spectrum

 

 

 

Figure 14: 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- IR spectrum

 

 

 

Figure 15: 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- Mass spectrum

 

CONCLUSION:

By this experiment it is concluded that both flower and leaf ethanolic extract of Pavetta crassicaulis Bremek. showed appreciable aphrodisiac activity but the major compounds 2-Tert-Butyl-4,6-Bis(3,5-Di-Tert-Butyl-4-Hydroxybenzyl) Phenol and 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-present in it were not responsible for the activity. The compound which is responsible for the activity should be investigated to confirm its traditional use.

 

AKNOWLEDGEMENT:

The authors thankful to Dr Vimarsh Gowda, University of Agricultural sciences, Bangalore, for suggestions and supportduring our experimental work.

 

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Received on 19.10.2017          Modified on 15.11.2017

Accepted on 20.12.2017       ©A&V Publications All right reserved