Studies of Phytochemicals, Antioxidant, and Antibacterial Activities of Pinus gerardiana and Pinus roxburghii Seed Extracts

Pine seeds are considered as nonwood forest products (NWFP) with regularly increasing market's demand. They can be eaten in various ways such as roasted or raw. In addition, they are included in various traditional dishes like in cookies, sauces, candies, cakes, breads, and other bakery items and, moreover, for medicinal purposes. GC-MS study is performed to analyze the phytochemical compounds present in the seed extracts of Pinus roxburghii (Chir) and Pinus gerardiana (Chilgoza). In total, 25 compounds were identified each in Chir and Chilgoza. In Chir seeds, abundantly present compounds were 2,4-di-tert-butylphenol (16.6%), followed by ç-Terpinene (9.9%) and cyclohexanol, 4-ethenyl-4-methyl-3-(1-methylethenyl)-, (1à,3à,4á) (9.8%), whereas in Chilgoza seeds, the maximum amount of compound was 1-hexyl-1-nitrocyclohexane (17.3%), followed by phenol, 2,6-bis(1,1-dimethylethyl) (15.4%), and heptadecane, 2-methyl (8.4%). The total phenolic content of Chir seed sample was 1536 ± 4.35 (mg GAE/100 g), whereas in the Chilgoza seed extract was 642.66 ± 2.08 (mg GAE/100 g). The application of RP-HPLC-DAD system revealed that Chir and Chilgoza seeds have maximum quantity of catechin (15.77 ± 0.16 μg/mg and 17.49 ± 0.32 μg/mg, respectively). Both Chir and Chilgoza seed extracts exhibited significant antioxidant (radical scavenging) potential, through H2O2 (618.94 ± 21.45 μg/mL and 575.16 ± 19.88 μg/mL) and DPPH (552.60 ± 13.03 μg/mL and 429.15 ± 3.80 μg/mL) assays, respectively. Additionally, a well-known antibacterial potential was also found in both plants' dichloromethane extracts, with 64 to 256 μg/mL of minimum inhibitory concentrations. As a whole, result shows the importance of both plants as a naturally occurring phytochemical source with significant antibacterial and antioxidant activity.


Introduction
Pine trees are one of the most common and widely grown species in the Himalayan region. Categorized under the Pinus genus and Pinaceae family, they are the largest conifer's families in the world and always remain evergreen woody conifer trees [1]. In the Himalayan region (HR), five species of pine are considered indigenous, distributed at different elevation such as Pinus gerardiana (Chilgoza), Pinus wallichiana (Kail), Pinus roxburghii (Chir), Pinus merkusii (Merkus), and Pinus kesiya syn. Insularis (khasi) as shown in Table 1 [2]. Among these, Chilgoza tree nuts have highly incomparable nutritious value therefore, commercialized for edible use and consumed in roasted form; these seeds are added as an ingredient in different dishes [3]. Whereas Chir trees are abundantly present and have ethno medicinal value, nuts are traditionally consumed in India and Pakistan [4,5].
P. roxburghii, known as Chir pine trees, is generally 55 m tall and over 100 cm diameter breadth height (dbh). Tree bark is thick in dark reddish-brown while winter buds are brown, ovoid, small, and nonresinous. The tree leaves are needle-shaped in flabellate-triangular arranged in 3 per bundle and cylindrical in cross-section. Cones are pedunculate, short, ovoid, range in 10-15 × 6-9 cm, while seeds are small, about 8-12 mm in length with a long wing of 25 mm, and normally ripen in April [6].
P. gerardiana is traditionally called Chilgoza trees to a medium height (17 to 27 m) and 2-4 m in dbh. The branches are small and horizontal with glabrous bark and silver grey [7]. Leaves are needle-like shaped and dark green, arranged three per cluster. However, the male cones are longer than female ones, which are ovoid with hard woody scales. These seeds are in dark brown colored, pointed from top and cylindrical, and normally ripen in October [8].
Data available to date demonstrate that these trees are composed of different types of phenolic compounds, at varying amounts depending on the geographical origin, harvesting time and storage, with distinct biological activities being also reported [9]. In past two decades, massive studies were carried out on essential oils and extracts of several parts of pine trees. Studies generally focused on nutritional value, chemical composition, food supplements, and in drug formulation [10]. The natural bioactive compounds, including phenols, terpenes, flavonoids, alkaloids, and saponin obtained from different Pinus spp., have reported for their potency against several diseases, e.g., asthma, diabetes, neurodegenerative diseases, cancer, oxidative stress-related diseases, cardiovascular-related problems, liver and kidney disorders, and various pathogenic infections [2]. Among the phytochemicals present, these pine nuts usually contain inherent antioxidants that help in reducing the oxidation rate, namely, flavonoids, e.g., flavonols, and flavanones in various glycoside and aglycone form [11]. For instance various phenolic group constituents like as catechin, gallic acid and quercetin show antioxidant, antiallergic, antimicrobial, anti-inflammatory, UV protection, and anticancer activity [12,13]. Ellagic acid has potency to inhibit the oxidation of low-density lipoprotein [14]. Vanillic acid is highly efficient to reduce oxidative stress and Aβ1-42-induced cogni-tive impairment, therefore very effective in Alzheimer's and other neuron-related disease [15].
Till now, studies have been carried out to assess the phytochemical composition and biological potential of Chilgoza seeds [3,16]. In case of P. roxburghii except seed, the chemical composition and biological activities have been studied on its various parts, viz., needles and bark [4]. In this sense, the present study is aimed at evaluating the total phenolic content of P. gerardiana and P. roxburghii seed extracts and their bioactive compounds and at investigating the antioxidant and antibacterial roles. In addition, chemical composition and quantification of phenolic compounds were assessed by GC-MS and HPLC-DAD.

Preparation of Seed Extracts.
At room temperature, deshelled Chilgoza and Chir samples were grounded into powder with the help of commercial mixer grinder. Powdered seed samples of 50 g were added to 500 mL conical flask containing 250 mL dichloromethane (DCM) solvent each and kept in incubator cum shaker for 48 h at 37°C. Each sample was strained through Whatman no. 1 filter paper. After the extraction process, the liquid extracts were collected and then concentrated at 40°C by using a rotary vacuum evaporator. The prepared extracts were collected and stored at 4°C in refrigerator for further analysis [17].
2.3. Total Phenolic Content. The total phenolic content (TPC) of Chilgoza and Chir was determined by using Folin-Ciocalteau reagent [18]. Seed extracts (about 20 μg) were separately taken and made volume up to 1 mL by 2 BioMed Research International adding distilled water. Then, Folin-phenol reagent (500 μL) was added into that, and 2.5 mL sodium carbonate Na 2 CO 3 (20%) was also added. It was mixed properly and kept away from light for 40 min for incubation and color development. Postincubation, the absorbance was taken at 725 nm. Gallic acid calibration curve was constructed, and linearity was found in 5-25 μg/mL range. Seed extract TPC was stated in mg of gallic acid equivalent (mgGAE/100 g seed extracts) by using the standard curve.  [20]. From the stock, 0.01 mL was taken and diluted up to 1 mL diluents to make necessary dilutions and filtered through 0.22 μ PES membrane filters and injected in HPLC system.

Antioxidant Activities
Scavenging Assay. The free radical scavenging potential of two seeds' extract was evaluated following the method followed by Bhatti et al. [21] with slightly modifications. Stock solutions of both seeds' extracts (1 mg) were prepared in methanol (MeOH), and further different methanolic solution concentrations (20-640 μg/mL) were prepared. From each concentration, 300 μL was added to methanolic solution 2700 μL of DPPH (4 mg/100 mL). The mixture solution was incubated in absence of light at 37°C room temperature for one hour. Free radical scavenging efficacy of extracts was based on the initial purple color disappearance. Absorbance of solution was taken at 517 nm. For positive control, ascorbic acid was used [21]. Scavenging capacity of DPPH was determined using the formula below given: where the IC 50 of DPPH radical was calculated from the line regression of the percentage of remaining DPPH radical against the sample concentration.
2.6.2. Hydrogen Peroxide Scavenging Assay (H 2 O 2 ). The capacity of seed extract to scavenge H 2 O 2 was estimated following the procedure of Bhatti et al. [21]. Briefly, 0.1 mL extract aliquots (20-640 μg/mL) were added into an Eppendorf tubes to made volume up to 0.4 mL with addition of 50 mM (pH 7.4) phosphate buffer and (2 mM) H 2 O 2 solution (0.6 mL). Mixture was properly vortexed and kept for 10 min, and then, absorbance was read at 230 nm. Ascorbic acid was used as positive control [21]. The extracts' ability to scavenge the H 2 O 2 was evaluated by using the following equation: where A 0 is the absorbance of blank and A 1 is the absorbance of sample.  [22]. DCM extracts were prepared in Mueller Hinton broth, and serial dilutions were obtained, ranging between 0.5 and 256 μg/mL. Bacteria (1-2 × 10 8 CFU/mL) were transferred to test tubes and incubated at 37°C for 24 h. Minimum inhibitory concentrations (MIC) were determined, being considered as the lowest concentrations without visible turbidity. Colistin was used as a positive antibacterial control, while DCM was used as negative control [22].
Hoon et al. also reported the highest quantity of catechin in Chilgoza DCM seed extract, but our data on EGCG contradict that obtained by this author [3]. Zulfqar Table 6. Ascorbic acid showed stronger antioxidant activities in both DPPH (326:70 ± 9:64 μg/mL) and H 2 O 2 (375:73 ± 11:73 μg/mL) assays as compared to both seed powder extracts tested. Comparing both extracts, the antioxidant potential of Chilgoza seed extracts in both DPPH and H 2 O 2 assays was higher compared to that of Chir seed   Antibiotic resistance modulation, antimicrobial, anti-Leishmania, antitumor, analgesic, antioxidant, anti-inflammatory, and antimalarial [34] ç-Terpinene Monoterpene Antibacterial [35] 9-Hexacosene Alkene Analgesic and anti-inflammatory [36] 1-Undecanol Aliphatic alcohol OH Bactericidal, larvicidal, and membrane-damaging activity [37] 1-Eicosanol Primary alcohols HO Antitumor and antibacterial activity [38] sample extract, which seems to be attributed to the presence of the high amount of antioxidant phytocompounds in Chilgoza samples as detected through the HPLC-DAD system, although the TPC in Chir seed samples was almost double than Chilgoza seeds. However, earlier studies reported on pine nuts' phytochemical composition analyzes the presence of tocopherols, carotenoids, phytosterols, linoleic acids, and vitamin C, all of them revealing strong antioxidant potential, being their concentration higher than that in phenolic compounds [3,9,25]. The results obtained by DPPH assay to Chilgoza seeds DPPH were found accordingly with Hoon et al., who reported that an IC 50 value of DCM seed extract was as good as compared to EtOAc, EtOH, HEX, and MeOH extracts. Moreover, Chilgoza water extract results were much better [3]. Zulfqar et al. reported that the percentage of DPPH inhibition of both MeOH and EtOAc extract of P. gerardiana dry nuts was 76:33 ± 2:51% and 73:67 ± 2:75% at concentration of 10 mg/mL and was statistically insignificant [16]. Valero-Galván et al. reported that methanol seed extract of P. cembroides grown in the five states of Mexico revealed different antioxidant activity assessed by DPPH assay [24]. P. halepinsis found in Palestine region displayed that methanolic extract by maceration and Soxhlet extraction method showed IC50 of 0.12 mg/mL and 0.43 mg/mL, respectively [10]. Mahdhi et al. studied maximum antioxidant activities from P. halepensis methanolic-aqueous seed extracts by using DPPH at concentration of 0.08 mg/mL [23]. Su et al. reported that Pinus koraiensis seed (PKS) ethanol extract displayed significant scavenging activity on 2,2-diphenylpicrylhydrazyl (DPPH) (EC50, 0:023 ± 0:004 mg/mL) and significant suppressive effect on lipid peroxidation in liver as well as enhance the glutathione (GSH) and superoxide dismutase (SOD) antioxidant enzyme levels and reduce malondialdehyde (MDA) content in the brain and liver of rat [27]. Stem Table 7. Colistin was used as a positive control (MIC value was 8 μg/mL).
Both Chilgoza and Chir seed antibacterial potentials were expected due to the occurrence of the antimicrobial compounds 3-carene, 2,4-di-tert-butylphenol, 1-hexyl-1nitrocyclohexane, naphthalene, α-pinene, γ-terpinene, 1undecanol, and 1-eicosanol, as reported in Tables 4 and 5. The possible target sites of phytocompounds in microbes are cell membrane, cell wall, and different enzymes. Salim et al. found that the antibacterial activities of P. halepinsis ethanolic seed extracts displayed good inhibition percentage against bacteria Staphylococcus aureus, E. coli, and Shigella at range of 0.02 g/mL [10]. Sharma et al. reported the antibacterial activity against Pseudomonas aeruginosa, K. pneumonia, and E. coli to the bark hydroalcoholic extract of three types of pine species, viz., P. roxburghii (Chir), P. wallichiana (kail), and P. gerardiana (Chilgoza) by well diffusion method, despite P. wallichiana displayed the most prominent antibacterial activity [40].

Conclusion and Future Perspectives
Secondary metabolites present in pine seed extracts are coated with excellent biological properties. In this study, the DCM seed extract of P. gerardiana and P. roxburghii revealed to be a rich source of molecules with interesting antioxidant and antimicrobial effects. The obtained results are positive and, if supported by in vivo studies, may be further proposed to be used for therapeutic purposes. In the  Other studies should also be done to a better understanding of the impact of seed collection from different regions with altitudinal variation, in addition to correlation with climate, soil, and regional geographic data in chemical composition. Equally important will be to perform a combined analysis of protein, amino acid, minerals, and lipid profiles to reach a more clear understanding on the real potentialities of these less investigated trees. However, the Chilgoza seeds are eaten in roasted form in several countries, but still Chilgoza and Chir seeds are not utilized in functional food development. Recently, our group has developed the cookies having Chilgoza and Chir seeds used in decorated form to enhance its nutritional value [41]. Still there is a need to develop functional/nutraceutical foods using Chilgoza and Chir seeds which ultimately gives new employment horizon to the hilly area people.
The limitation of the current study is the selection of extraction solvent. We believe that DCM solvent has not much compatibility with our seed samples; that was the reason we got antioxidants and antibacterial activity at higher concentration.

Data Availability
The data used to support the findings of this study are available from the corresponding authors from request.

Conflicts of Interest
The authors have no conflicts of interest to declare.