INTRODUCTION:

Herbal medicines have gained appreciation today since much scientific research has been conducted to prove the therapeutic effects and efficacy of plant-derived pharmaceutical medicines against various diseases. Safety and effectiveness become issues that make herbal medicines and traditional medicinal preparations preferable to synthetic drugs^1,2,3. Validating and screening active ingredients such as plant metabolites and phenolic compounds contained in parts of plants are demanded to initiate the discovery of herbal medicines.

The use of plants in traditional healing practice is widely recognized as one of Indonesian culture. Many ethnobotanical studies have been carried out to reveal the traditional use and knowledge of indigenous communities in Indonesia. The Enclave Cikaniki area is part of The Cikaniki Resort of Gunung Halimun Salak National Park (GHSNP) in Western Java, whose communities are crucially influenced by natural resources from the surrounding forest. The traditional use and knowledge of medicinal forest plants by Sundanese communities living in the enclave have been practiced since the settlement was established in the Dutch East Indies era⁴. The practice and significant benefits of the plants as traditional medicines become a legacy passed down from generation to generation. The latest survey in 2021 reported that at least 112 medicinal plant species were used by the community in the Cikaniki area⁴. However, the potential of the chemical compounds in some plants need to be screened, identified, and developed, particularly the species that have been used traditionally in the communities for a long time.

Index Cultural Significance (ICS) analysis showed that among the local medicinal plants used in the Cikaniki area, Staurogyne elongata (Nees) Kuntze, Blumea balsamifera (L.) DC., Plantago major L., Polygala paniculata L., Turpinia montana (Blume) Kurz, Goniothalamus macrophyllus (Blume) Zoll., Lophatherum gracile Brongn., Scleria levis Retz., Begonia robusta Blume, Heptapleurum spp., and Liquidambar excelsa (Noronha) Oken., possess significant value in the communities⁴. Staurogyne elongata which is endemic to Sumatra and Java was known and used as edible medicinal plant in Western Java only^5-8. Its composition, potential antioxidant and antibacterial activities have been reported in few research^{9, 10}. Blumea blasamifera^11-13, P. major^14-16, P. paniculata^17-19, L. gracile^20-22, and G. macrophyllus^23-25 are remarkable medicinal plants in many parts of the world which have been observed both in vitro and in vivo related to chemical composition and bioactivities.

Polygala paniculata and P. major are not native to Java²⁶. However, they had been naturalized and used in the communities as medicinal plants. Liquidamber excelsa is a typical fresh vegetable for montane communities in Western Java. However, studies of medicinal use and bioactivities of L. excelsa were limited to anti-inflammatory and stamina boosters²⁷ and anticancer for oral cancer^28,29. The use of T. montana^30-31, S. levis^32,33, B. robusta^5,34, and Heptapleurum spp.³⁵ as traditional medicine has been recorded but have limited information on phytochemical and pharmaceutical activities.

The ethnobotanical surveys had been conducted on the local communities living in and surrounding GHSNP since 1995 and recorded lists of medicinal plants they used^4,5,36,37. However, the phytochemistry and pharmaceutical activities of some medicinal plants used in the communities are still limited or never observed. Therefore, our study aimed to investigate the bioactive components, antioxidant, antimicrobial activities, and the nutritional composition from leaves of selected medicinal plants with high ICS values from former ethnobotanical study.

MATERIALS AND METHODS:

Materials:

Five hamlets in the enclave of Cikaniki Resort, GHSNP were visited to learn firsthand and collect the cultural data of the local community's various species of plants used in traditional medicine. They are Citalahab Sentral, Citalahab Bedeng, Citalahab Kampung, Cilanggar and Garung which administratively belong to Malasari Village of Bogor Regency, West Java. The topography of the five hamlets is hilly, with altitudes ranging from 950-1100 m.a.s.l and climate type A. The annual rainfall ranges between 3200-6000 mm, and the annual temperature is between 16-30°C³⁸. Based on the direct field observation, light intensity ranges from 662-19300 lux. Plant materials were collected from natural forests that relatively close to the settlements.

Cultural Data Collection through Index Cultural Significance (ICS)

Based on interviews with 57 respondents from five hamlets of the Cikaniki Resort, the information was converted into three components: quality (q), intensity (i), and exclusivity (e) value. The conversion value is then calculated using the ICS formula³⁹.

The calculation results for medicinal plant criteria are sorted from the highest to the lowest. About 13 species with the highest ICS value were selected to identify the species name before it's collected for phytochemical screening. The species identifications were assisted by previous publications of plant inventories from GHSNP area^{5, 36, 40} and through the Plants of The World Online (POWO) website. Thirteen GHSNP plants were used as samples: S. elongata, L. excelsa, B. robusta, B. balsamifera, P. major, G. macrophyllus, S. levis, T. Montana, L. gracile, P. paniculata, H. fastigiatum, H. polybotryum, and H. scandens.

Samples preparation and extractions:

The leaves were collected randomly from several trees in the Cikaniki Resort and thoroughly mixed. The pre-processed leaves were placed on a clean newspaper and allowed to air dry at room temperature. Thus, the leaves are dried in an oven at 40 °C and mashed into powder. Sample extraction for phytochemical screening and biological activities was carried out using the Lailaty et al.⁴¹ method with modification. Samples were macerated on a shaker by each solvent with a ratio of 1:5 for 24 hours. Variation solvents used were aquadest and ethanol P.A. Filtrate were separated by Whatman No. 1 filter paper. The filtrate was concentrated by rotary evaporator. Next, the extracts were stored at 4 °C for further use.

Qualitative Phytochemical Screening:

Identification of alkaloids from thirteen GHSNP plant extracts used tree reagents test. The positive alkaloids with different precipitate forms: a brown to black (Bouchardat), a white or yellow lump (Mayer), an orange-brown (Dragendorf)^42,43. In flavonoids identification⁴², we used cat whisker leaves as standard solutions. The other qualitative phytochemical screening had been conducted to find the existence of tannins⁴³, saponins⁴² and terpenoids⁴² compounds.

Quantitative Phytochemical Screening:

a) Determination of Total Phenol:

One mg extract was dissolved in 1ml of methanol. The standard solution of quercetin was made in duplicate with concentrations of 10, 20, 30, 50ppm. Each sample was taken 250µL, put into 3 different test tubes. The blank methanol solution was added with 2.2 ml of aquadest. The standard solution of quercetin was added with aquadest according to the concentration. The sample extract was added with 2.45 ml of aquadest to each tube. Furthermore, the blank solution, standard solution, and sample were added 150 µL of 5% NaNO₂, shaken, and allowed to stand for 2 minutes. Then, 150 µL of the blank solution, standard solution, and sample were added with 150 µL of 10% AlCl₃, shaken, and allowed to stand for 8 minutes. The blank, standard, and sample were added with 2 ml of 1 M NaOH. Measurement of the absorbance of each sample using a spectrophotometer with a wavelength of 510nm.

b) Determination of Total Flavonoid:

One mg extract was dissolved in 1 ml of methanol. The standard solution of quercetin was made in duplicate with concentrations of 10, 20, 30, 50 ppm. Each sample was taken 250 µL, put into 3 different test tubes. The blank methanol solution was added with 2.2 ml of aquadest. The standard solution of quercetin was added with aquadest according to the concentration. The sample extract was added with 2.45 ml of aquadest to each tube. Furthermore, the blank solution, standard solution, and sample were added 150 µL of 5% NaNO₂, shaken, and allowed to stand for 2 minutes. Then, 150 µL of the blank solution, standard solution, and sample were added with 150 µL of 10% AlCl₃, shaken, and allowed to stand for 8 minutes. The blank, standard, and sample were added with 2 ml of 1 M NaOH. Measurement of the absorbance of each sample using a spectrophotometer with a wavelength of 510 nm.

Determination of Antioxidant Activity:

The extract solution in 2 ml of methanol was added to a solution of 0.5 ml DPPH (1 mM in methanol). The mixture was shaken and allowed to stand at room temperature for 30 minutes. The resulting absorption was measured at a wavelength of 515 nm. The percentage of sample inhibition was calculated based on the difference in absorption between the blank (A0) and the sample (As)⁴⁴.

DPPH-scavenging effect (%) = [1-(As/A0)×100]

The percentage of DPPH attenuation activity is plotted against the sample concentration. The 50% (IC₅₀) attenuation value is calculated from the attenuation percentage on the sample concentration graph. The test was repeated two times, and quercetin was used as a comparison. Sample preparation for testing used several concentrations (10, 50, 100, 200, 500 µg/mL).

Determination of Antibacterial Activity:

The antimicrobial test was carried out using the disk diffusion method on a double-layer test medium⁴⁵. Mueller Hinton Agar (MHA) media was used for the bottom layer with an agar concentration of 20 g/L. The same medium was used for the upper layer with an agar concentration of 8 g/L each. The test bacteria were grown on Mueller Hinton Broth (MHB) media and incubated at 37 ^oC overnight in a shaking incubator. The test microorganism culture was then diluted to 106 CFU/mL⁴⁶ using a semi-solid MHA growth medium as the top layer, then poured over the bottom layer media. The sample is placed on top of the medium after the medium containing the test microorganism has solidified. The agar plate media was then incubated at 4 ^oC for 1 hour and continued at 37 ^oC for two days. The test was carried out in three repetitions. Samples that produce an inhibition zone on the test medium are considered positive for having antibacterial activity.

Screening of Metabolite Compounds by GC-MS:

The ethanol extracts of each selected species were analyzed for the secondary metabolite profile using Gas Chromatography-Mass Spectrometry (GC-MS) merk Agilent 7890B (GC) and 5977A (MSD). The metabolites were identified for their group compound based on online databases, i.e. Pubchem (https://pubchem.ncbi.nlm.nih.gov/), Kyoto Encyclopedia of Genes and Genomes (KEGG), ChemSpider (http://www.chemspider.com/), LIPID MAPS (http://www.lipidmaps.org/), and Metabolomics Workbench (https://www.metabolomicsworkbench.org/data/).

Data Analysis:

The qualitative data were analyzed descriptively, and the quantitative data were analyzed using One-Way ANOVA with Fisher's LSD (Least Squares Difference) when the result was significant. Meanwhile, the antioxidant activity of selected medicinal plants was analyzed using MANOVA. The Anova and Manova analysis were executed using Minitab 19.

RESULT:

Local Medicinal Plants Used by GHSNP Community:

There are 122 species from 54 families of plants used as medicinal plants in the GHSNP community⁴. The commonly used families come from the Asteraceae, Zingiberaceae, Apocynaceae, Acanthaceae, and Arecaceae. However, many of the species used are introduced herbs. Thus, in carrying out further quantitative and qualitative phytochemical tests, it is necessary to rescreen to select the native species that are most widely used by the community (Table 1).

Table 1: ICS value of thirteen species of medicinal plants used by community in GHSNP

Scientific name	Family	Vernacular name	Habitus	Habit	Hamlets					ICS	Descriptions
					Cs	Cb	Ck	Cl	Gr
Staurogyne elongata (Nees) Kuntze	Acanthaceae	Rendeu	Herb	Wild	√	√	√	√	√	351	Part Used: Leaves
											Mode of Preparation: drink the water from boiled plants
											Diseases: Kidney, liver, postpartum
Blumea balsamifera (L.) DC.	Asteraceae	Sembung	Shrub	Wild, cultivated	√	√	√	√	√	228	Part Used: Leaves, Roots, Stems
											Mode of Preparation: drink the water from boiled plants
											Diseases: Postpartum, ulcer, bloating, soreness, skin, respiration
Plantago major L.	Plantaginaceae	Ki urat	Herb	Wild	√	√	√	√	-	162	Part Used: Leaves, Roots
											Mode of Preparation: pounded and smeared, eat/direct use, drink the water from boiled plants
											Diseases: Eyes
Polygala paniculata L.	Polygalaceae	Ki Kumat	Herb	Wild	√	-	√	√	√	121.5	Part Used: Leaves, stems, all part of plant
											Mode of Preparation: drink the water from boiled plants, dried plant and then the simplicial boiled to drink
											Diseases: Cough, soreness, eyes
Turpinia montana (Blume) Kurz	Staphyleaceae	Karas tulang	Tree	Wild, cultivated	-	√	√	√	√	112.5	Part Used: Stems, roots, all part of the plant
											Mode of Preparation: drink the water from boiled plants
											Diseases: Stomachache, diarrhea, fever, postpartum
Goniothalamus macrophyllus (Blume) Zoll.	Annonaceae	Ki cantung	Tree	Wild	-	-	√	√	√	105	Part Used: Stems, bark, roots, all part of the plant
											Mode of Preparation: drink the water from boiled plants
											Diseases: Wounds, coughs, hypertency
Lophatherum gracile Brongn.	Poaceae	Tangkur gunung	Herb	Wild	√	√	√	√	-	94.5	Part Used: Leaves, mixed roots and tubers
											Mode of Preparation: dried plant and then the simplicial boiled to drink, drink the water from boiled plants
											Diseases: Male stamina, strengthen teeth, ulcers, back pain, postpartum
Scleria levis Retz.	Cyperaceae	Rumput ilat	Herb	Wild	-	-	-	√	-	84	Part Used: Umbut (the middle part of the young stem), tubers
											Mode of Preparation: baked
											Diseases: Wounds, postpartum, sore throat
Begonia robusta Blume	Begoniaceae	Hariang	Herb	Wild	√	√	-	√	-	72	Part Used: Stems
											Mode of Preparation: eat/direct use
											Diseases: Back pain, relieve sore throat
Heptapleurum fastigiatum (Miq.) Seem.	Araliaceae	Ki kunti	Woody climber	Wild	-	-	√	√	-	72	Part Used: leaves, water from the liana stems
											Mode of Preparation: eat/direct use
											Diseases: Eyes, kidney, cough, sore throat, postpartum
Heptapleurum polybotryum (Miq.) Seem.	Araliaceae	Ki kunti	Woody climber	Wild	-	-	√	√	-	72	Part Used: leaves, water from the liana stems
											Mode of Preparation: eat/direct use
											Diseases: Eyes, kidney, cough, sore throat, postpartum
Heptapleurum scandens (Blume) Seem.	Araliaceae	Ki kunti	Woody climber	Wild	-	-	√	√	-	72	Part Used: leaves, water from the liana stems
											Mode of Preparation: eat/direct use
											Diseases: Eyes, kidney, cough, sore throat, postpartum
Liquidambar excelsa (Noronha) Oken	Altingiaceae	Rasamala	Tree	Wild, cultivated	√	√	-	√	-	36	Part Used: Leaves
											Mode of Preparation: eat/direct use
											Diseases: Fever

Notes: ICS = Index Cultural Significance, Cs = Citalahab Sentral, Cb = Citalahab Bedeng, Ck = Citalahap Kampung, Cl = Cilanggar, Gr = Garung, √ = found in the hamlet

Table 2: Phytochemical screening of thirteen species of GHSNP

Species	Part Used	Alkaloids			Flavonoids	Tannins	Terpenoids	Saponins
Species	Part Used	Bouchardat	Mayer	Dragendorf	Flavonoids	Tannins	Terpenoids	Saponins
S. elongata	Leaves	+	-	+	-	-	+++	++
L. excelsa	Leaves	+	-	+	-	+++	++	+++
B. robusta	Roots	+	-	+	-	-	-	+
B. balsamifera	Leaves	+	-	+	-	+	+	++
P. major	Leaves	+	-	+	-	-	-	++
P. major	Roots	+	-	+	-	-	-	++
G. macrophyllus	Leaves	+	-	+	-	+	+	++
S. levis	Leaves	+	-	+	-	+	-	+
T. montana	Leaves	+	-	+	-	++	-	+
L. gracile	Roots	+	-	+	-	-	-	+
	Stem	+	-	+	-	-	-	++
	Leaves	+	-	+	-	-	-	++
P. paniculata	Leaves	+	-	+	-	+++	++	+++
H. fastigiatum	Leaves	+	-	+	-	++	+	+++
H. polybotryum	Leaves	+	-	+	-	+	++	++
H. scandens	Leaves	+	-	+	-	++	+++	+++
Positive Reactions		Brown to black	White or yellow lump	Orange-brown	Red color	Green-violet	Red turning blue-green	Foam

Notes: + : extract was containing phytochemical compounds, - : extract was not containing phytochemical compounds

Figure 1. DPPH scavenging activity of seven species from GHSNP, a) aquadest extracts, b) ethanolic extracts

Total Phenol and Flavonoid Content:

In this study, the total phenolic content expressed as gallic acid equivalent (%) in the P. major extract was found to be highest (32.15±2.68%), not significant with T. montana (30.59±2.72%). Table 3 shows that the total phenol content (TPC) is higher than the total flavonoid content (TFC). The results in Table 4, L. excelsa extract shows the highest total flavonoid content, significantly different from P. major and G. macrophyllus (p<0.05).

Table 3: Total phenol and total flavonoid contents of seven species from GHSNP

Species	% Total Phenol Content ± SD	% Total Flavonoid Content ± SD
S. elongata	10.22 ± 0.97^cd	6.13 ± 0.08^e
L. gracile	3.79 ± 0.32e	8.17 ± 0.02^d
H. fastigiatum	13.19 ± 1.65^bc	6.43 ± 0.14^e
T. montana	30.59 ± 2.72^a	9.07 ± 0.12^c
L. excelsa	14.44 ± 1.89^b	11.85 ± 0.12^a
P. major	32.15 ± 2.68^a	10.53 ± 0.11^b
G. macrophyllus	7.41 ± 0.32^d	10.25 ± 0.60^b

Antioxidant activity:

The percentage of inhibition increased as the concentration of the extract increased (Figure 1). A relatively high increase in inhibition was found in P. major both in distilled water and ethanol extract. Meanwhile, The IC₅₀ value indicates that the ethanol extract is better than the aquadest extract. On the other hand, a lower IC₅₀ value indicating higher antioxidant activity was found in H. fastigiatum and G. macrophyllus in aquadest extract, also S. elongata in ethanol extract (Table 4).

Table 4: Determination of the antioxidant activity from seven species of leaf extracts by DPPH free radical scavenging assay

Species	IC50 ± SD (µg/mL)
Species	Aq extracts	EtOH extracts
Quersetin	5.17 ± 0.01	5.17 ± 0.01
S. elongata	486.78 ± 3.49	127.49 ± 0.15
L. excelsa	258.91 ± 0.40	128.48 ± 0.00
P. major	93.26 ± 0.52	288.26 ± 0.15
G. macrophyllus	42.43 ± 0.43	284.23 ± 0.01
T. montana	191.65 ± 1.45	138.42 ± 0.15
L. gracile	275,71 ± 2,95	220,33 ± 1,09
H. fastigiatum	14.24 ± 0.05	541.31 ± 0.52

Table 5: Statistical analysis for antioxidant activity

Parameters	F value	P value (<5%)
Solvent (S)	22928.42	0.000*
Concentration of extract (CE)	812882.44	0.000*
Plant species (P)	43226.96	0.000*
S*CE	3216.04	0.000*
S*P	58099.58	0.000*
CE*P	9105.32	0.000*
SCEP	10529.49	0.000*

IC₅₀ expresses the amount or concentration of extracts needed to scavenge 50% of the free radicals. The value of IC₅₀ is inversely proportional to the scavenging activity of the leaf extract. Table 4 shows the antioxidant activity of plants leaves extracted with aquadest and ethanol. The results show that the IC₅₀ value of different extracts varied significantly, ranging from 14.24 to 486.78 (µg/mL). A lower IC₅₀ value indicating higher antioxidant activity was found in Heptopleurum fastigiatum and G. macrophyllus in aquadest extract also S. elongata in ethanol extract. The aquadest extract of H. fastigiatum gave the lowest IC₅₀ value 14.24±0.05. H. fastigiatum leaf extract can neutralize free radicals maximally, but not as good as antioxidant quercetin (IC₅₀ = 5.17). This is due to quercetin's content, a pure antioxidant compound that can potentially neutralize the free radicals better than plant extracts. Based on statistical analysis, the parameters of solvent, extract concentration, plant species, and interaction between the parameters were significant (p<0.05) (Table 5).

Antibacterial activity:

The disk diffusion assay conducted the antibacterial analysis of the plant leaf extracts. The antibacterial susceptibility test of the aquadest and ethanolic leaf extracts from the seven species of GHSNP showed varying degrees of antibacterial activities for the selected bacterial species. Based on Table 6, it was shown that ethanol extract had higher antibacterial activity than aquadest extract, indicated by negative results in all species. The observation of the inhibition zone showed that the highest value was found in Turpinia montana. This indicates the high level of bioactive compounds that act as antibacterials based on the results of GC-MS.

Metabolite profiling by GC-MS:

The GC-MS analysis of the ethanolic extracts showed a wide chemical diversity, including 79 compounds for five species. The compounds were distributed by fatty acids, ketone, carboxylic acid, ester, furan, hydrocarbon, sesquiterpene, diterpene, and other organic compounds. They include organochlorine, calmore, glycerolipids, siloxane, organosilicon, amino, silane, and benzene. Fatty acids represent the main class of identified compounds presenting the main components of myristic acid, palmitic acid, oleic acid, valeric acid, and stearic acid. T. montana had the highest number of compounds (49 compounds). On the other hand, P. major is the lowest (21 compounds). The observed phytochemical compounds in these ethanolic extracts have been reported to possess biological activities and medicinal importance (Table 7).

Table 7: Biological activity from the compounds of ethanolic leaf extracts.

Compounds	Molecular weight	Formula	Total content of compounds (%)							Biological potency
Compounds	Molecular weight	Formula	Tm		Se		Pm	Lg	Le	Biological potency
Sesquiterpene
(-)-Globulol	222.37	C₁₅H₂₆O	0.809	-			-	-	-	Antimicrobial activity⁴⁷
Alpha.-Cadinol	222.37	C₁₅H₂₆O	1.031	-			-	-	-	Antimicrobial and antioxidant, ACE-inhibitory⁴⁸
Spathulenol	220.35	C₁₅H₂₄O	0.472	-			-	-	-	Antioxidant, anti-inflammatory, antiproliferative and antimycobacterial⁴⁹
Longifolene	204.35	C₁₅H₂₄	0.525	-			-	-	-	Natural autoxidation & anti termite, anti-inflammatory, antimicrobial^50-52
Gamma-Muurolene	204.35	C₁₅H₂₄	1.322	-			-	-	-	Anti-inflammatory and antinociceptive, antimicrobial and antioxidant⁵³
Trans-Calamenene	202.33	C₁₅H₂₂	0.747	-			-	-	-	Antimicrobia, cytotoxic^{54, 55}
Diterpene
Phytol	296.5	C₂₀H₄₀O	-	0.301			-	-	-	Plant metabolite, a schistosomicide drug and an algal metabolite⁵⁶
Terpenophenols
THCA-A	386.643	C₂₄H₃₈O₂Si	0.690	-			-	-	-	Psychoactive⁵⁷
Fatty acid
Oleic Acid	282.5	C₁₈H₃₄O₂	-	0.122		-		-	-	An Escherichia coli metabolite, a plant metabolite, a Daphnia galeata metabolite, an antioxidant and a mouse metabolite, flavoring agent, acaricides, herbicides, insecticides, Plant Growth Regulators⁵⁸
Valeric acid	174.31	C₈H₁₈O₂Si	1.126	0.705		1.048		4.029	-	Plant metabolite, alzheimer treatments, dementia syndrome^59-60
Stearic acid	356.7	C₂₁H₄₄O₂Si	4.256	6.828		4.702		4.231	7.791	Personal care, teeth whitening and antibacterial⁶¹
Siloxane
Tetracosamethyl-cyclododecasiloxane	889.8	C₂₄H₇₂O₁₂Si₁₂	2.614	-			-	-	-	Cytotoxic and antimicrobial⁶²
Octadecamethyl-cyclononasiloxane	667.4	C₁₈H₅₄O₉Si₉	1.112	-			-	-	-	Antibacterial⁶³
Ester
Decyl oleate	422.7	C₂₈H₅₄O₂	-	-			-	0.518	-	Personal care, emollient, skin conditioning, softener and conditioner⁶⁴
Organoclorine
Methylene chloride	84.93	CH₂Cl₂	1.172	1.776			1.836	8.273	6.999	Ingredients of insecticides/pest/food additives detox, addiclenz, and additox⁶⁵
Alkane
Pentadecane	212.41	C₁₅H₃₂	-	-			0.792	-	-	Flavouring, fragrance⁶⁶

Notes: Tm=Turpinia montana; Se= Staurogyne elongata; Pm= Plantago major; Lg= Lophatherum gracile; Le= Liquidambar excelsa.

DISCUSSION:

The use of local plants as medicines is usually passed down from generation to generation in the community of Cikaniki Resort of Gunung Halimun Salak National Park (GHSNP)⁴. Medicinal plants are considered necessary because they can help people maintain stamina and body health. Medicinal plants are generally planted in yards close to villages or residences, in line with various studies on the diversity of medicinal plant species in home gardens, providing many conveniences when using them and disseminating information about traditional knowledge⁶⁷. Therefore, the habitus used as medicine is commonly shrubs and herbs. However, medicinal plants also live in their natural habitat or the edge forest near the village. Medicinal plants from part of trees are usually taken directly from forests and are rarely planted in home gardens.

Ethnopharmacological studies are important in drug discovery because a number of therapeutic products have been developed using ethnobotany knowledge. Phytochemicals from medicinal plants are extensively studied because those majority contain a variety of bioactive compounds with health benefits⁶⁸. Based on phytochemical test, the thirteen selected plants showed the presence of different types of active constituents. The alkaloid screening was carried out using three different tests. The Bouchardat and Dragendorf tests were positive for all the species examined, meanwhile, the Mayer test was negative. The Mayer's test negative result was expected due to the limit of nitrogen and ion of potassium (K⁺) from potassium tetraiodomercurte (II) reaction that makes the complex of potassium-alkaloid precipitation cannot be formed⁶⁹. Notably, all extracts were tested negative for flavonoids in qualitative phytochemical test. The samples' low concentration of flavonoid content could not be detected in the qualitative analysis⁷⁰. Meanwhile, the tannins and terpenoids in some of the examined species and saponins are all present. Based on the availability of the secondary metabolites, other estimations of the plant's vital constituents viz. total phenols and flavonoids, were further determined. Qualitative phytochemical screening will support the explanation of a wide range of chemical compounds produced by plants, meanwhile quantification of those metabolites will assist in the extraction, purification, and identification of bioactive compounds⁷¹.

Phenolic compounds are regarded as essential antioxidants. They are widely distributed among various plant species. Based on Anwar et al.⁷², L. excelsa has major components identiﬁed were monoterpenes (-pinene, β-pinene, -phellandrene, limonene, and β-phellandrene), phenolic acid (3,4-dihydroxy benzoic acid, gallic acid), and flavonoid (apigenin, kaempferol, and quercetin). Kaempferol and quercetin in L. excelsa have intense anticancer activity in human oral tongue cancer SP-C1. Previous studies by Sakanaka et al.⁷³ have reported the importance of phenolic compounds in scavenging free radicals. As a result, plant extracts' total phenolic and flavonoid contents are frequently used to explain their antioxidant activities. Flavonoid antioxidants are primarily secondary metabolites produced by plants to repel herbivorous predators, competitors, also attract pollinators⁷⁴. Determining the antioxidant activity studies on different plant species can reveal this species' value as a source of new antioxidant compounds⁷⁵. In reality, there are over a hundred extracts that claim to have antioxidant properties. Such evidence is required to lend scientific credibility to folklore uses of traditional medicines while also being supportive of prospective medicines and treatments⁷⁶.

Secondary metabolites found in plants include tannins, terpenoids, alkaloids, and flavonoids, which have antimicrobial properties⁷⁷. Our results showed that the ethanol crude extract of T. montana was more effective than some other plant extracts due to its inhibitory effects on S. aureus, with an inhibition zone value of 0.7 cm. This result is like previous research that has shown that the ability of plant extracts against bacteria depends on the solubility of the bioactive constituents⁷⁸. On the other hand, the lower inhibition zone in other extracts might be since the plant extracts, being in crude form, contain smaller concentrations of bioactive compounds⁷⁹. The type of solvent and the extraction procedure used have a large impact on the successful prediction of botanical compounds from plant material. Furthermore, the environmental and climatic conditions of the plants influenced the level of antimicrobial activity⁸⁰. Furthermore, the ethanolic extracts of S. elongata and L. gracile showed an antibacterial response to both gram-positive and gram-negative bacteria.

The presence of various compounds with variable chemical structures was revealed by gas chromatography and mass spectroscopy analysis⁸¹. From our GC-MS observation, methylene chloride and stearic acid were found in five species. Pentanoic acid is a plant metabolite expressed in all species, except L. excelsa. Oxalic acid; eicosane, 1-iodo-; and protocatechoic acid is only found in L. excelsa. The compounds of 3-(2-Ethyl-piperidin-1-ylmethyl)-8a-methyl-5-methylene-decahydro-naphtho[2,3-b]furan-2-one; nordihydrocodeine; and pentadecane are specific found in P. major extract. Nazarizadeh⁸² also reported that P. major has phenols (ferulic acid), flavonoids, and tannins have the highest amount in Plantago leaves than seeds. Sebacic acid, 4 methylhept-3-yl undecyl ester; 3-Amino-2-piperidone, 2TMS derivative; 1,3-Dioxolane, 4-ethyl-5-octyl-2,2-bis(trifluoromethyl)-, trans-, is only owned by L. gracile. Meanwhile, phytol and oleic acid are only found in S. elongata. These compounds are usually used as plant metabolites and drug materials.

In recent years, combined GC-MS has emerged as a critical tool in natural product research. Individual components in a complex mixture, particularly oil, can be effectively resolved using gas chromatography⁸³. Plant product formation is influenced by both intrinsic and extrinsic factors, resulting in qualitative and quantitative changes in chemical composition. The comparison is sometimes so dissimilar that the active constituents of one species are completely absent in the same species of plant collected from different locations, rendering it completely unsuitable for the intended purpose⁸⁴. Sesquiterpene is the most common compound found in T. montana with beneficial properties. According to Cox-Georgian et al.⁸⁵, terpenes are antimicrobial agents, and their mode of action is the weakening of tissue and the cell wall of the microorganisms. They also serve as anticancer and antidiabetic agents. Turpinia montana extract contains several sesquiterpenes, namely (-)-globulol, alpha-cadinol, spathulenol, longifolene, gamma-muurolene, and trans-calamenene as antimicrobial and antioxidants. THCA-A (Tetrahydrocannabinolic acid – A) is also found in T. montana. This compound has psychotropic activity, also found as bioactive compounds in Cannabis. It is obvious that the leaf extracts contain a variety of phytocompounds that may have suitable biological properties for use in the pharmaceutical industry. However, additional research, such as bioprospecting, is required to support its biological properties⁸⁶. Furthermore, studies must be conducted to generate relevant evidence in order to understand and establish the pharmacokinetic and pharmacodynamic actions of compounds isolated from GHSNP plants.

CONCLUSION:

Thirteen native species from Gunung Halimun Salak National Park have the potential to be utilized and developed as alternative medicines. Staurogyne elongata has the highest ICS value and can inhibit the growth of S. aureus and Escherichia coli. Plantago major had the highest total phenol and flavonoid content, not significantly different from T. montana. G. macrophyllus and T. montana had the highest antioxidant activity from both extracts. From the GC-MS analysis, sesquiterpene is the most common compound found in T. montana leaf extract with health benefits, especially for antioxidant and antimicrobial properties.

CONFLICT OF INTEREST:

The authors confirm that we do not have any conflict of interest in this submission.

ACKNOWLEDGMENTS:

The authors thanked Gunung Halimun Salak National Park for the support and the plant samples used in this research. We also acknowledge the facilities, scientific and technical support from Advanced Characterization Laboratories Serpong and Indonesian Culture Collection (InaCC) Laboratories, National Research and Innovation Agency through E-Layanan Sains, Badan Riset dan Inovasi Nasional.

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Received on 14.03.2023 Modified on 17.09.2023

Research J. Pharm. and Tech 2024; 17(5):2121-2132.

DOI: 10.52711/0974-360X.2024.00336

Species	Extracts	Antibacterial activity		Diameter of Inhibition Zone (cm)
Species	Extracts	*E. coli*	*S. aureus*	*E. coli*	*S. aureus*
P. major	Aquadest	-	-	-	-
P. major	Ethanol	-	+	-	0.3
T. montana	Aquadest	-	-	-	-
T. montana	Ethanol	-	+	-	0.6
S. elongata	Aquadest	-	-	-	-
S. elongata	Ethanol	+	+	0.1	0.1
L. excelsa	Aquadest	-	-	-	-
L. excelsa	Ethanol	-	+	-	0.2
L. gracile	Aquadest	-	-	-	-
L. gracile	Ethanol	+	+	0.1	0.1