Chemical composition, larvicidal and antioxidant activities of latex from Garcinia morella (Gaertn.) Desr.

Abstract

The present study revealed that the latex of Garcinia morella (Gaertn.) possessed 204.27 mg/g phenolic acid and 124.92 mg/g of flavonoid content along with other phytochemicals. Gas chromatography-mass spectrometry (GC–MS) revealed the presence of 20 compounds in the latex, with the major compounds identified as 5-Oxohexanenitrile (18.7%), phenol, 2,4-bis(1,1-dimethylethyl)- (24.64%) and Hexadecanoic acid (22.85%). The latex showed toxicity against treated third instar larvae of Culex quinquefasciatus with LC₅₀ and LC₉₀ values of 132.54 and 483.15 ppm. The latex also showed significant antioxidant activities with EC₅₀ values of 205.5 µg/ml in the 1,1-diphenyl-2-picrylhydrazyl (DPPH), 95.53 µg/ml in phosphomolybdate, 308.1 µg/ml in hydrogen peroxide (H₂O₂) scavenging assays.

Introduction

Increasing number of morbidity and mortality in humans due to mosquito borne diseases has inflicted great economic loss in not only tropical and subtropical countries but also throughout the world. Like any other biting flies, mosquitoes transmit many diseases like malaria, dengue, yellow fever, filariasis and encephalitis, which under severe conditions can even cause death. WHO has declared mosquitoes as “Public Enemy Number One” (WHO 1996). Prevention of mosquito borne diseases can be made possible only by preventing the proliferation of mosquitoes. Use of insecticides and synthetic pesticides such as organochlorine and organophosphate compounds has given considerable success due to their speedy action and easy application. Due to the increasing resistance of mosquitoes and environmental damage caused by the synthetic products (Brown 1986; Russell et al. 2009), alternate pest management strategies like biological insecticides have been developed (Mehlhorn et al. 2005).

Culex quinquefasciatus, a tropicopolitan mosquito transmits zoonotic disease which affects humans and animals and causes avian malaria, lymphatic filariasis, St. Louis encephalitis, Western equine encephalitis and West Nile fever. Bancroftian filariasis, caused by Wuchereria bancrofti and transmitted by Culex quinquefasciatus, accounts for the 95% of lymphatic filariasis in India (Michael et al. 1996). India accounts for 40% of total global prevalence of filariasis infection in world (Ramaiah et al. 2000).

Garcinia morella (Gaertn.) Desr. popularly known as Indian Gamboge, is native to Western Ghats of India and is also found in Sri Lanka and Indo-China Himalayan region. The plant is a multipurpose tree. The extract and resin gum finds numerous ethno medicinal uses for curing wound rot, carbuncle, ulcer, abdominal pains, constipation, spleen enlargement and sore. It is also used in paint and lacquer industries. The fruit rinds are used as condiments and garnish. The bioactive compounds like morellin and gambogic acid isolated from the fruits and bark have been evaluated for their antibiotic and anticancer properties (Subba Rao et al. 1978; Tang et al. 2011). Other active compounds like Isomorellin, Desoxymorellin, Dihydoisomorellin, morellic acid, Isomorellic acid, Morellinol and Dihydromorelloflavone have also been reported. Due to its high economic value, various strategies have been employed in crop improvement for commercial production of this plant (Joseph et al. 2014).

To date, there are no reports on larvicidal activity of latex isolated from G. morella against filariasis causing vector C. quinquefasciatus. In view of recent development in usage, increasing interest and demand for biological insecticides as an alternate source for synthetic compounds, this study was undertaken to assess the potential larvicidal activity of latex obtained from G. morella against filariasis causing vector C. quinquefasciatus, and its antioxidant activities.

Materials and methods

Plant material

Healthy and mature trees of G. morella growing near Sirsi, India (14°33′6.4″N, 74°32′45.4″E) were used as a source of fresh latex. The latex of the G. morella was collected by making small incisions on the bark in small tubes (10 ml) containing acetone to produce 3:1 mixture. The mixture was filtered using muslin cloth to remove any debris present in the latex. The filtered mixture was concentrated in a rotary vacuum evaporator and stored at 4 °C until further bioassay.

Chemical composition

The latex was qualitatively analyzed for various phytochemicals according to the method of Guevara (2005). The gas chromatography–mass spectrometric (GC–MS) analysis of latex was conducted by a Shimadzu GC–MS (model QP2010S). Compounds were identified by direct comparison with the retention times and mass spectral data with those in the National Institute of Standards and Technology 11 (NIST 11) library.

Larvicidal activity assay

The C. quinquefasciatus eggrafts were collected from the drainage near railway station of Dharwad, India and reared in laboratory (29 ± 3 °C, 75–85% RH, Photoperiod of 14 h light and 10 h dark). The larvae were fed with dog biscuit/Brewer’s yeast (3:1). The larvicidal activity was carried out as per the standard procedure recommended by WHO (2005). The latex was dissolved in 1 ml of acetone and different concentration, i.e. 37.5, 75, 150, 300 and 600 ppm, and was prepared using dechlorinated tap water. Control was setup by 1 ml of acetone in 249 ml of water. Twenty five late third instar larvae were used for larvicidal assay and for each concentration 5 replicates were maintained. After 24 h of exposure, larval mortality was calculated and the lethal concentration LC₅₀ and LC₉₀were calculated by probit analysis.

Antioxidant assays

The antioxidant assays were carried out to determine the free radical scavenging activity by DPPH (1,1-diphenyl-2-picrylhydrazyl), total antioxidant activity (TAC) by phosphomolybdate assay, hydrogen peroxide scavenging activity (H₂O₂) and reducing power assays according to standard methods (Blois 1958; Prieto et al. 1999; Ruch et al. 1989; Oyaizu 1986). Ascorbic acid (AS) was used as standard.

Results and discussion

Latex is a complex emulsion of various phytochemicals and other constituents like gum, resins and secondary metabolites. It is produced from the laticiferous cells of plants as a defense mechanism against herbivorous insects such as inhibition of egg hatching, larval growth and moratlity (Ramos et al. 2006). The latex of G. morella showed the presence of various phytochemicals like terpenoids, flavonoids, saponins, tannins, cardiac glycosides, and phenols. Phenolic acids (204.27 mg Gallic acid equivalent/g) and flavonoids (124.92 mg Quercetin equivalent/g) were the major components present in the latex followed by alkaloids (19.27 mg/g) and tannins (1.66 mg/g). Table 1 and Fig. 1 represent the constituents present in the latex obtained by GC–MS analysis. A total of 20 compounds were identified from the latex. The major constituents in the latex were 5-Oxohexanenitrile (18.7%), phenol, 2,4-bis(1,1-dimethylethyl)- (24.64%) and Hexadecanoic acid (22.85%).

Table 1.

Chemical constituents of latex from Garcinia morella

Sl. no.	Retention time	Percentage concentration	Compounds
1	16.147	18.7	5-oxohexanenitrile
2	19.724	24.64	Phenol, 2,4-bis(1,1-dimethylethyl)-
3	23.656	1.25	Cis-sesquisabinene hydrate
4	24.147	1.04	Octadecane
5	26.417	3.03	3-heptadecanol
6	26.664	0.95	Octadecanal
7	26.795	0.85	Isopropyl myristate
8	28.464	1.2	7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione
9	28.857	0.59	Hexadecanoic acid, methyl ester
10	29.369	1.32	3-ethyl-3-undecanol #
11	30.117	2.07	9,17-octadecadienal, (z)-
12	30.261	5.44	9-octadecenal, (z)-
13	30.75	5.36	Octadecanal
14	32.604	2.12	Eicosane
15	34.469	1.15	Heptadecanal
16	36.956	1.04	Celidoniol, deoxy-
17	38.572	4.22	Octadecanoic acid, 3-oxo-, methyl ester
18	39.216	22.85	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester
19	39.626	0.66	Tetratetracontane
20	41.853	1.5	4-(2-tert-butyl-5-oxo-1,3-dioxolan-4-yl)butylformamide

Fig. 1.

GC-MS chromatogram of Garcinia morella latex

The characteristic activity of the extracts in plants is mainly due to the active compounds present in it. The C. quinquefasciatus third instar larval mortality after 24 h of treatment with latex at different concentrations is shown in Table 2 along with regression equation. The latex of G. morella showed LC₅₀ value of 132.54 ppm and LC₉₀ value of 483.15 ppm. Considerable mortality was observed against C. quinquefasciatus third instar larvae with increase in concentration, which was supported by the regression equation. By taking dosage (X) as independent variable and mortality (Y) as dependable variable, the regression equation showed that Y is positively related with X suggesting that the mortality increases with increased in dosage. The larval mortality at 75 ppm was 33%, which increase to 97% at 600 ppm.

Table 2.

Probit values of G. morella plant latex against larvae of Culex quinquefasciatus

Concentration (ppm)	Percentage of mortality ± SD	LC50 (ppm)	95% Fiducial limits (LCL-UCL)	LC90 (ppm)	95% fiducial limits (LCL-UCL)	Regression equation
37.5	10 ± 0.33	132.54	116.03–151.01	483.15	394.13–627.80	Y = 2.2815X + 0.1578
75	33 ± 0.57
150	49 ± 0.33
300	74 ± 0.33
600	97 ± 0.57

SE standard error (all values are mean of four replicates), LC ₅₀ lethal concentration that killed 50% of the exposed larvae, LC ₉₀ lethal concentration that killed 90% of the exposed larvae, LCL lower confidence limit, UCL upper confidence limit, Y mortality rate (significant at P < 0.05 level), X concentration (significant at P < 0.05 level)

Our results show the potentiality of G. morella latex and its constituents against filariasis causing vector Culex quinquefasciatus. In our study, the larvicidal activity was dose dependent and was notably similar to other reports on various plant latexes. Phytochemicals such as xanthones, alkaloids, steroids, phenolics and essential oils from different parts of the plant have been previously reported to have insecticidal activities (Shaalan et al. 2005). An alicyclic polyalcohol, d-pinitol isolated from the Acacia nilotica showed toxicity against Aedes aegypti and C. quinquefasciatus fourth instar larvae (Chaubal et al. 2005). Similarly, Rahuman et al. (2000) observed larvicidal activity of n-hexadecanoic acid extracted from the dried leaves of Feronia limonia against fourth instar larvae of Ae. aegypti, C. quinquefasciatus and Anopheles stephensi. The leaf extracts Argemone mexicana showed larvicidal activity against C. quinquefasciatus and an enhanced activity, when mixed with Clausena dentate (Sakthivadivel et al. 2012). The plant latex isolated Plumeria rubra, Calotropis procera and Euphorbia milli also showed larvicidal activity against various mosquito larvae during various developmental stages (Patil et al. 2012; Ramos et al. 2006; Shahi et al. 2010; Filho and Paumgartten 2000).

The electron donation ability of natural compounds can be measured by the bleaching of the purple colored solution by DPPH radical. The scavenging effect of latex on DDPH radical is as shown in Fig. 2a. The EC₅₀ values of the scavenging DPPH radical for latex is 205.5 µg/ml, which is higher compared to the standard ascorbic acid (198.8 µg/ml). The high antioxidant properties of the latex can be attributed to the presence of high amount of phytochemical compounds (phenolic hydroxyls) that are capable of donating hydrogen to free radicals to scavenge the potential change. The phospomolybdate assay is a quantitative method for estimation of total antioxidant activity (TAC) and is expressed as ascorbic acid equivalents. The latex showed strong antioxidant activity in dose dependent manner at concentration 100–500 µg/ml (Fig. 2b). The EC₅₀ value of antioxidant capacity of latex (95.53 µg/ml) is more pronounced than the standard ascorbic acid (97.09 µg/ml). The strong antioxidant activity of the latex can also be attributed to the presence of high amount of phenolic compounds. However, recent studies have shown flavonoids and related polyphenols also to contribute significantly to the phospomolybdate scavenging activity (Sharififar et al. 2009). The scavenging activity of H₂O₂ was concentration dependent as shown Fig. 2c. H₂O₂ occurs naturally at low concentrations in living organisms. H₂O₂ usually present is decomposed to water and oxygen and can release hydroxyl radicals which can cause DNA damage and initiate lipid peroxidation (Sahreen et al. 2011). The latex displayed strong antioxidant activity (EC₅₀ 308.1 µg/ml) compared to that of standard ascorbic acid (281.5 µg/ml). In reducing power assay, the yellow color of the solution changes to green depending upon the reducing power of the sample. The reductants present in the solution causes the reduction of Fe³⁺ to Fe²⁺, which is monitored by the absorbance at 700 nm. Reducing properties have shown to exert antioxidant activity by donating H atom to break free radical chain (Gordon 1990). Figure 2d shows the dose response curve for the reducing powers of G. morella plant latex. The reducing power increased with increase in concentration of sample. The highest reducing power of 1.90 ± 0.05 was observed at 500 µg/ml. The antioxidants present in the latex of G. morella caused the reduction of Fe³⁺ to Fe²⁺, thus proving reducing power.

Fig. 2.

Antioxidant activity of G. morella plant latex at various concentrations. Each value represents a mean ± SD (n = 3). a DPPH radical scavenging activity, b total antioxidant capacity, c H₂O₂ radical scavenging activity, d reducing power assay. AS ascorbic acid, % percentage

In conclusion, the finding in the present study suggests that the latex and active principles of G. morella may be explored as a potential source for natural larvicide. However, further investigations on its activity on non-target organisms and field trials are necessary. The results also showed that the latex is a good source for antioxidants due to the presence of phenolic and flavonoid contents and a good antimicrobial agent against food borne pathogens and can be used for isolation and preparation of natural antibiotics.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.