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Analysis of the chemical composition of root essential oil from Indian sarsaparilla (Hemidesmus indicus) and its application as an ecofriendly insecticide and pharmacological agent

The Indian sarsaparilla (Hemidesmus indicus) is a commonly used plant in Indian traditional medicine of Ayurveda for the preparation of various non-alcoholic beverages. However, limited studies are available on the essential oil of H. indicus roots (HRO); therefore, the study evaluated the antioxidant, anti-inflammatory and antidiabetic activities of H. indicus root essential oil as well as insecticide potential against the common pests of stored food materials (Sitophilus oryzae, Callosobruchus maculatus and Tribolium castaneum). The repellant efficacy of HRO was found to be high against S. oryzae (8.21 ± 0.55 μg/mL). Likewise, the fumigant potential was also observed for HRO against these pests; the higher activities were observed against S. oryzae and C. maculatus (32.46 ± 1.42 and 35.18 ± 1.62 μg/L). Besides, the essential oil was also found to be active as a contact poison, however, against all the three pests, the toxicity was above 100 μg/mm3, being the highest against C. maculatus (122.8 ± 3.57 μg/mm3). To analyze the possible effect of the essential oil on grains, the different grains were allowed to germinate and compared to that of normal; thus, the non-toxic nature of HRO against the stored products is also confirmed. The essential oil shown to have DPPH hydrogen peroxide and ABTS radical scavenging, nitric oxide scavenging potential, and inhibition of lipoxgenase, alpha-amylase and alpha-glucosidase. Overall, the present study concludes that the H. indicus may be a suitable repellant and fumigant agent against different pests of stored products and a possible antioxidant, anti-inflammatory, and anti-diabetic agent.


Introduction
Pests are animals that are having harmful effects directly or indirectly on crops, livestock, or humans. Among the different pests, insects are thought to be one of the most important organisms with pest status. Pests can cause serious loss to the economy of a country by reducing the productivity of various crops (Hernandez Nopsa et al., 2015). Apart from these, the particular group of insect pests that feed on the stored food products are more damaging, as they damage the stored food materials (Kumar and Kalita, 2017). Cereals and pulses are the important products that supply food materials to the whole world and the pests acting on them are therefore causing a serious threat to food safety (Yaseen et al., 2019). The most common pests of the stored grains include Sitophilus oryzae, S. zeamais, Tribolium sp., and Callosobruchus sp that feed on grains and other pulses. Fumigation with various pesticides is the currently employed strategy for the management of these pests; the commonly used pesticides include phosphine, sulfuryl fluoride, and pyrethrins (Huang and Subramanyam, 2005;Jagadeesan et al., 2018). However, the use of fumigant pesticides are known to cause deleterious effects to humans also; the exposure can induce toxic effects on the central nervous system, various types of cancers and acute poisoning often results in life-threatening conditions (Alavanja et al., 2014;Park et al., 2020;Van de Sijpe et al., 2020). This intensified the requirement for new pesticide compounds that are having significant repellency and fumigant potential to the different pests and are less toxic to humans.
Plant-derived essential oils are emerging as key pesticide molecules that are having less impact on the environment as well as on human/animal/plant health (Al Dawsari Mona, 2020;Zaka et al., 2019). Among the different essential oils produced, those produced from edible plants or plant parts are in the limelight of becoming an eco-friendly biocide. Apocynaceae family is one of the widely studied family for their biological, toxicological, pharmacological, ethno-medicinal, and botanical values (Bhadane et al., 2018). Several plants belonging to the group are evolved as promising insecticidal, larvicidal and antimicrobial drug candidates (Dhileepan, 2014;Panneerselvam et al., 2013). Among these plants, the Indian sarsaparilla (Hemidesmus indicus) is utilized as an aromatic agent in a variety of food and beverages (Nandy et al., 2020). The plant is reported to be beneficial against a variety of disease conditions by virtue of its antioxidant and anti-inflammatory potentials (Desai et al., 2017;Kundu and Mitra, 2013). Further, the plant is found to reduce secondary diabetic complications by inhibiting the polyol pathway enzyme aldose reductase (Haroon et al., 2021). Anticancer properties are also attributed to the plant against colon (Turrini et al., 2019), breast (Bansod et al., 2021) and lung cancer cells (Pathiranage et al., 2020). However, limited studies are available on the essential oil of H. indicus; a previous study has identified the bioactive component 2-hydroxy-4-methoxy benzaldehyde (Jirovetz et al., 2002). It is, therefore, necessary to evaluate the potential of the essential oil as a possible biocide against different pests of stored products including Sitophilus oryzae, Callosobruchus maculatus and Tribolium castaneum. Further, the essential oil was also evaluated for their anti-radical effects, antidiabetic effects, and toxic/adverse effects in terms of the germination potential of grains.

Plant materials collection and essential oil extraction
Hemidesmus indicus roots were collected from naturally growing areas in the Calicut district, Kerala, India. The roots were then cleaned to remove soil, washed in cold water and powdered. The essential oil was extracted by steam distillation in three independent isolations; the yield and composition were compared. The preparation was according to the modified methods of Nagarajan et al. (2001); briefly, about 250 g of the freshly powdered roots of H. indicus was weighed and transferred to a steam distiller (6 h). The organic essential oil layer was then isolated and concentrated before storage at À20°C.

Chemical composition analysis by GC/MS
Chemical composition was analyzed using a Varian CP 3800 GC/ MS system with a column size of 30 m Â 0.25 mm inner diameter and a thickness of 0.25 lm. The analysis conditions were as follows; the oven temperature of 50°C (120 s), temperature rise (2°C/min till 250°C), the temperature of injection port and detector was 250°C. The helium was used as the carrier gas at a flow rate of 1 mL/min. The compound identification was carried out by comparing the retention index (RI) with the NIST library.
2.4. Inhibition of alpha-amylase and alpha-glucosidase inhibition 2.4.1. a-Amylase inhibition activity Different concentrations (0-100 lg/mL) of essential oil were mixed with 400 lg/mL of porcine a-amylase solution in 20 mM phosphate buffer (pH 6.9) and incubated at 25°C for 15 min. After that, about 0.5 mL of starch solution (0.8%) was added and again incubated for 15 min. The enzyme activity was stopped by adding 1.5 mL of 3,5-dinitrosalicylic acid (DNS) reagent. The solution was then made up to 10 mL using double distilled water and optical density was measured at 540 nm. Percentage inhibition was estimated by comparing the values with a control.

a-Glucosidase inhibition assay
About 0.1 mL of the a-glucosidase enzyme solution (25 lg/mL) was mixed with different concentrations of essential oil and incubated for 10 min at room temperature. Further, 0.05 mL of pnitrophenyl-a-D-Glucoside (10 mM) was added and again incubated at 35°C for 30 min. At the end of incubation, 0.1 mL of sodium carbonate (w/v) solution was used to stop the enzyme activity and the optical density was recorded at 405 nm in a spectrophotometer.
2.5. Efficacy of H. indicus root essential oil as a biopesticide 2.5.1. Fumigant toxicity The fumigant potential of the essential oil was analyzed against the different pests according to the method described below; briefly, the H. indicus root essential oil was dissolved in acetone and then coated on a Whatman No.1 filter paper having a diameter of 2 mm at their different concentrations (0-100 mg/L air). The filter papers were then dried to remove the traces of acetone and then placed inside a glass bottle. To each of such glass bottles, 20 adult pests were released and then bottle as properly closed. The pests were observed continuously for 72 h and mortality was recorded. The LC50 value was calculated by plotting the mortality against respective doses.

Repellant activity
The repellency of H. indicus essential oil was determined according to the methods described by Kłyś et al. (2020) using a set of insect breeding containers. Briefly, the essential oil at different concentrations was dissolved in acetone and then on a Whatman no. 1 filter paper disc having a diameter of 8 mm. The disc was placed in the perforated (1.5 mm) inside the container along with wheat grains (5 g) and 20 adult insects. The repellency was estimated as the number of insects moving away from the inside container through the perforation to the outside. The median repellant dose (RD 50) was determined by plotting the essential oil concentration against the repellency at each concentration.

Contact toxicity
The contact toxicity of essential oil was estimated according to the methods described by Patiño-Bayona et al. (2021). The different concentrations of essential oil were applied topically using a micro-syringe on the prothorax region of adult insects (10 nos/ treatment group) and the treated insects were then kept in a glass bottle. A set of insects were kept as untreated control; the toxicity up to 48 h was recorded for the calculation of LD50 value.

Effect of essential oil on germination potential of grains
The phytotoxic potential of the essential oil was determined according to the methods of Liu et al. (2006). Three different concentrations of essential oil were prepared (100, 250 and 500 mg/ mL) and approximately 5 mL of these was transferred to Petri dishes containing filter paper and seeds (30 seeds). The dishes were then sealed and incubated at 25 ± 1°C for 48 h. The seal was removed and the plates were allowed to remain at room temperature for 6 days and readings were taken every 24 h. The percentage germination in each dose and negative control was estimated.

Statistical analysis
The essential oil isolation was conducted in three independent extractions and they were compared for chemical constituents. The result of biocide studies was expressed as Mean ± SD of five independent analyses.

Yield and chemical composition analysis by GCMS
The average yield of essential oil by steam distillation from the root of H. indicus was about 1.28 ± 0.11%. The predominant compound that was present in the plant was 2-hydroxy-4-methoxy benzaldehyde (92.3%); apart from that, nerolidol and camphor were the other important compounds that were present in the essential oil (Table 1).

Antioxidant effects, anti-inflammatory and anti-diabetic potentials
The antioxidant potential of the essential oil was estimated as radical scavenging and ferric reducing powers (Fig. 1). The IC50 values of DPPH radical scavenging and ABTS scavenging assays were 51.38 ± 0.39 and 48.94 ± 0.64 mg/mL. The hydrogen peroxide scavenging potential was found to be 64.93 ± 0.59 mg/mL (Fig. 1a).
Anti-inflammatory effects were estimated in terms of nitric oxide scavenging with IC50 value 88.83 ± 1.21 mg/mL and lipoxygenase inhibitory potential was 75.48 ± 0.78 mg/mL (Fig. 1b). Alphaglucosidase and alpha-amylase inhibition was observed with IC50 values 44.71 ± 1.55 and 50.89 ± 0.86 mg/mL (Fig. 1c). Table 1 The percentage composition of root essential oil of Hemidesmus indicus isolated by steam distillation; composition was determined by GC-MS analysis.

Compounds
Retention Index

Repellency, fumigant and contact toxicity of H. indicus essential oil
The pesticide property of H. indicus root essential oil was evaluated in terms of the repellency potential, fumigant toxicity and contact toxicity. The repellent effect was high against the S. oryzae, followed by C. maculatus and T. castaneum with respectively IC50 values 6.08 ± 0.26, 10.16 ± 0.63, and 7.59 ± 0.28 mg/L of air (Table 2). Likewise, the fumigant potential of the essential oil was high against S. oryzae (22.6 ± 0.87 mg/L of air). On the contrary, the contact toxicity was high against C. maculatus (78.2 ± 4.58 mg/mg).

Phytotoxicity of essential oil on germinating seeds
Pesticides are not usually devoid of secondary effects on the grains and pulses. Therefore, the phytotoxic effect of the essential oil against the wheat grains was evaluated. However, there observed no significant toxicity in terms of germination of wheat grains in the root essential oil of H. indicus for 144 h (Table 3).

Discussion
The Hemidesmus indicus commonly known as Indian sarsaparilla is a shrub widely used in dietary beverages; the roots of the plant are also used in the traditional medicines as decoctions. Despite their wide usage and product range, limited reports were available on the essential oil of H. indicus. The chromatographic analysis indicated the presence of nerolidol and camphor, and to a significantly higher concentration, 2-hydroxy-4-methoxy benzaldehyde. Previous studies by Jirovetz et al. (2002) also indicated a similar pattern of GCMS and increased level of 2-hydroxy-4methoxy benzaldehyde in H. indicus root essential oil.
The antioxidant potential of the essential oil was identified in terms of the multiple radical scavenging abilities. The free radicals are thought to initiate various cellular reactions that are causing damages to macromolecules like proteins, enzymes, cell membrane lipids and even nuclear materials (Alara et al., 2019). Hence, the scavenging of these radicals often protects the cells from various noxious insults. Inhibition of nitric oxide and lipoxygenase is also important in terms of the inflammatory reduction ability of the essential oil (Alzarea et al., 2021). Together, the antioxidant and anti-inflammatory properties of H. indicus essential oil makes it a possible pharmacological agent. Inhibition of carbohydrate metabolic enzymes a-amylase and a-glucosidase contribute to its anti-diabetic properties; therefore, intake of H. indicus essential oil may also help to regulate the body glucose levels (Patel and Ghane, 2021).
Among the different groups of insects, numerous species are known to be deleterious to humans in terms of health and economy. Vectors of diseases are generally considered deleterious to humans and plants; however, the pests, which are known to cause serious damages to the crops and products are also causing significant decline with respect to the productivity and thereby the economic status of a country. The pests of stored products are a major threat as they are usually causing damages to the preserved materials. The results of the study indicated the possible use of essential oil from H. indicus as a repellant against the S. oryzae, C. maculatus, and T. castaneum, the common pests of stored products. Further, the essential oil has also been found to be efficient to induce mortality in pests as a fumigant and contact insecticide. Previous studies have also indicated the possible role of essential oil derived volatile compounds as possible pesticide candidates against various pests of stored grains (Singh et al., 2021). It is also reported that benzaldehyde and its derivatives (the predominant compound of the essential oil is 2-hydroxy-4-methoxy benzaldehyde) are known for their efficacy as antimicrobial and insecticidal compounds (Ullah et al., 2015). It is therefore possible that the bioactive compound of H. indicus essential oil is responsible for its biocidal properties; further purification of the compound may yield a novel insecticidal compound.
Pesticides are often known to cause significant environmental toxicants and are also deleterious to human health. Previous studies have indicated that fumigant pesticides like phosphine and methyl bromide often induce neurotoxicity and also induced leukaemia in humans (Alavanja et al., 2014;Park et al., 2020;Van de Sijpe et al., 2020). It was also known to cause significant phytotoxicity mediated through genotoxic effects on plants (Fatma et al., 2018). However, the root essential oil of H. indicus is found to be safer and no sign of toxicity was observed in the germinating grains was observed during the study.
The study thus concludes that the root essential oil of H. indicus is a novel repellent and insecticidal compound against the pests of stored products. They can be utilized to minimize the effect of pest-induced damages to the crops and economy to an extent. Further, the non-toxic nature of the essential oil makes it an ecofriendly option to control these pests.

Conclusion
The Hemidesmus indicus root essential oil was found to be a strong antioxidant agent by inhibiting various types of free radicals. In addition, the anti-inflammatory potential and antidiabetic effects of the essential oil increases its pharmacological values. Further, it is an effective biocide against the different pests of stored food materials. The fumigant potential oil of the essential oil enhances its application even in food storage houses. Apart from that, there observed no significant variation between the germination potential of grains exposed to the different doses of the essential oil. It is thus possible that H. indicus root essential oil may be an effective and eco-friendly biocide against different pests of stored grains.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Project No: RSP 2021/11. The authors are also thankful to DBT-STAR scheme and RUSA grants for the infrastructural development support.