INTRODUCTION:

Medicinal plants contain traditional bioactive compounds beneficial to humans in curing various diseases¹. Medicinal plants have been used since ancient times² as a tonic, food, or to increase immunity and body strength^2–5. It has the characteristic of a thin and low rosette^6,7. Green leaves with toothed linear and curved growth and white flowers with elongated tubular ⁸. The plant belongs to Campanulaceae and spreads in tropical, temperate, and cold regions^9,10.

The flower and leaf parts of H. longiflora (L.) G. Don is commonly used in medicine and food. The ethanol extract of H. longiflora (L.) G. Don leaves alkaloids, flavonoids, polyphenols, monoterpenoids, sesquiterpenoids, quinine, steroids, coumarins, tannins, and saponins^11–13. The compound (−)-Lobeline is known that have been isolated from Lobelia inflata, extensively studied its structure and biological activity¹⁴, and is thought to have many functional similarities with the genus Hippobroma¹⁵.

Bioactive compounds in H. longiflora (L.) G. Don flower extract is a group of fatty acids and their derivatives¹⁶.

Pharmacologically, the flavonoid content of H longiflora (L.) G. Don can be used as a natural sunscreen and inhibit the development of cataracts^17–19. Other uses of H. longiflora (L.) G. Don is also an antioxidant, anti-cancer, anti-inflammatory, antibacterial, anti-diabetic, antimalarial, anti-tumor, antimicrobial, antifungal, anti-insecticide, and antiseptic^20–22. Other studies prove that the bioactive constituents of H. longiflora (L.) G. Don can be used as a folk remedy for bronchitis, laryngitis, asthma, wounds, eye medication, anti-neoplastic, hemostasis, and analgesic^23,24.

Recent reports state that a 75% concentration of ethanol extract from H. longiflora (L.) G. Don leaves effectively inhibit the growth of Staphylococcus aureus and Salmonella typhi with robust criteria^25,26. Other studies also mentioned that ethanol extract from H. longiflora (L.) G. Don leaves inhibits corneal neovascularization after seven-day administration to the eyes that have experienced chemical trauma due to the pesticide Rotraz© 200EC²⁷. H. longiflora (L.) G. Don is documented in Indonesian pharmacology as one of the traditional herbs (JAMU) by the Indonesian population. They are traditionally extracted by the natives to treat human diseases. However, the complete constituents of this plant are still unclear. Hence, this study aims to compare the phytochemical constituents of H. longiflora (L.) G. Don (leaves and flowers) with GC-MS analysis and further evaluate the potential for biological activity.

MATERIALS AND METHODS:

Plant Materials:

Fresh plants of H. longiflora (L.) G. Don a were collected from farmers' gardens in Mustika Jaya District, Bekasi, West Java, Indonesia (6°17'31"S, 107°1'46"E) in August 2022. This plant is authenticated at ELSA, Indonesia Botani Identification Services, and specimens are deposited at the university with voucher number 1961537. The sample was washed and stored at 20^oC until subsequent analysis.

Sample Extraction Preparation:

Each part was weighed by 100g and oven dried at 33.5^oC for 45 minutes following procedure ²⁸. Dried samples were mashed with a grinder and stored at -18°C in low-density polyethylene bags²⁹. For three days, the stock concentration of 20mg/mL extract was prepared by soaking a powder sample of 20g of powder (40 mesh) in Pro Analyst ethanol (99.8%). The solution mixture is stirred and placed in a 40°C waterbath and shaken at 250rpm for 1hour³⁰. The mixture was centrifuged (2000rpm) for 10min, followed by filtration. The residue was immersed in a solvent and evaporated at 65^oC using Rotary Evaporator. The extraction results are stored inside the fume hood to evaporate the excess solvent further until it produces a stickier and more concentrated extract. The extract was held at 4°C until further analysis³¹.

GC-MS Condition:

Gas Chromatography-Mass Spectrometry (GC-MS) was used to decompose the phytoconstituents of target plants ³². A combination of a gas chromatograph 7890 A (Agilent 19091-433HP, USA) and a mass spectrophotometer equipped with an HP-5 MS molten silica column connected to a 5675C-MS-D and a Three Axis detector was implemented³³. The capillary column of GC-MS HP-5MS (30m 0.25mm ID 0.25m) consists of 5% diphenyl and 95% Dimethylpolysiloxane²⁸. The ethanol extract was filtered using a 5μL filter in split mode (8:1). Helium gas was used as the carrier at a rate of 1.2mL/min at an injector temperature of 250°C. Then the analyte is separated into a silica capillary column. The oven program is set up as follows: an initial temperature of 80°C which is directly raised by 3°C/min to 150°C and held for 1 minute, then increased by 20°C/min to 280°C and held for 26minutes²⁸. Ionization energy (70eV) was used to determine the mass spectrum.

Data Analysis:

The Agilent Mass Hunter software analyzes bioactive compounds by comparing mass fragments and the standard mass spectrum. Biological activity data were analyzed using the PubChem library database, Chemistry WebBook, and FOODB from the Metabolomics Innovation Center. This library was also used in Guang-Mei Tang's research in 2022³⁴.

RESULTS AND DISCUSSION:

This study shows that the GC-MS method is a powerful combination of phytoconstituents composition analysis ^5,35–37. The components can be separated effectively by GC and MS completing thorough component identification^12,38–41. We agree that the GC-MS method provides an excellent opportunity to explore phytocomponents as has been done by many previous researchers^{26,28,50,42–49}. This study used the GC-MS to determine the phytoconstituents of H. longiflora (L.) G. Don leaf and flower extracts. The GC-MS chromatogram of H. longiflora (L.) G. Don recorded a total of 37 peaks corresponding to phytochemical compounds recognized by attributing Retention Time (RT) and Concentration (%). As a result, as many as 16 unique metabolite compounds were present in the leaf and 21 in the flower of H. longiflora. The results of the leaf chromatogram of H. longiflora (L.) G. Don showed the highest RT value in Squalen from the Prenol lipids group (Figure 1).

-3,7,11,Trimethyl-dodeca-2,4,6,10-Tetraenal- is a unique compound that is only present on leaves with two different time retention. This lipid prenol derivative is a product of lycopene oxidation ⁵¹ in biological tissues and has been studied to have a role as an anti-depressant. Another unique compound detected that also has two retention times is 12-Methoxy-18-Nor-5.Beta.-Podocarpa-8,11,13-Trien-3.Alpha. O.L., a derivative of the Hydrophenanthrenes group, has yet to be discovered in terms of its benefits. Interpretation of the mass spectral chromatogram of H. longiflora (L.) G. Don compounds is presented in Table 1.

Figure 1: Chromatogram of H. longiflora (L.) G. Don leaf

Table 1: List of compounds leaf and flower of H. longiflora (L.) G. Don

M.F.	Compound	M.W.	Classification	Leaf		Flower
M.F.	Compound	M.W.	Classification	R.T.	% of Area	R.T.	% of Area
C₇H₆O	Benzaldehyde	106.12	Benzene and substituted derivatives	-	-	5.866	1,2
C₂₀H₄₀O	(2E)-3,7,11,15--Tetramethyl-2-Hexadecene--1-OL	296.5	Prenol lipids	31.296	4,17	-	-
C₂₆H₄₃N₅O₄	(9E)-9-Octadecenoic Acid	489.7	Fatty acyls	31.585	1,17	-	-
C₁₅H₁₆O₂S	12-Methoxy--18-Nor-5.Beta. --Podocarpa-8,11,13-Trien-3.Alpha.-OL	260.4	Hydrophenanthrenes	-	-	37.370	1,89
C₁₅H₁₆O₂S		260.4	Hydrophenanthrenes	-	-	38.260	2,62
C₆H₅COCH₃	1-Phenylethanone	120.15	Organooxygen compounds	8.155	1,65	-	-
C₁₈H₃₈	2,6,10-Trimethyl, - 14-Ethylene-14-Pentadecne	278.5	terpenoid	29.399	5,64	29.399	1,12
C₁₅H₁6O₂S	2,4',6-Trimethyldiphenylsulfone	260.4	Not Found	37.784	14,81	-	-
C₃₀H₅₀	2,6,10,14,18,22--Tetracosahexaen, 2,6,10,15,19,23--hexamethyl-,(all-E)-	410.7	Prenol lipids	36.529	39,01	36.515	7,54
C₉H₁₀N₂O₄	2-[4-(-1 Methylethoxy) bnztl]-4,6-pyrimidinediol	210.19	Benzene and substituted derivatives	37.895	1,5	-	-
C₁₈H₁₂O₂	2-Hydroxy-2-Phenyl-1-Acenapthenon	260.29	Naphthalenes	37.322	1,85	-	-
C₁₅H₂₂O	3,7,11, -Trimethyl-dodeca-2,4,6,10-tetraenal	218.33	Prenol lipids	29.613	1,24	-	-
C₁₅H₂₂O	3,7,11, -Trimethyl-dodeca-2,4,6,10-tetraenal	218.33	Prenol lipids	29.758	1,82	-	-
C₃₀H₄₈O	4,4,6a,6b,8a,11,12,14b,Octamethy-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-Octadehydro-2H-Picen-3-one-	424.7	Prenol lipids	-	-	49.272	2,27
C₁₉H₂₂O₆	6,7,8,9-Tetrahydro--3-Methoxymethyl-5,9-Dimethyl-8-Hydroxy-Naptho [1,2-B] Furan	346.4	Naphthofurans	38.067	2,34	-	-
C₁₇H₁₂N₂O	6H-Pyrido- [4,3-B] Carbazole-5-Carboxaldehyde, 11-Methyl-	260.29	Carbazoles	-	-	38.032	41,3
C₁₀H₁₈	8-Methyl--7-Thiabicyclo [4.2.1] nonan	138.25	Not Found	-	-	43.018	1,33
C₃₉H₇₀O₆	9,12-Octadecadienoic Acid	635.0	Fatty Acyls	-	-	36.143	1,08
C₁₀H₂₁NO	Acteamine, N-(1,1,3,3-tetramethyl butyl)-	171.28	Indoles and derivatives	-	-	38.825	38.825
C₈H₈O	Acetophenone	120.15	Organooxygen compounds	-	-	8.127	5,04
C₃₀H₅₀O	Alpha Amyrin	426.7	Prenol lipids	48.375	2,23	-	-
C₃₁H₆₄	Hentriacontane	436.8	Saturated hydrocarbons	-	-	40.701	3,87
C₂₀H₄₂	Hexadecane, 2,6,10,14-Tetramethyl-	282.5	Diterpenes	-	-	42.873	2,45
C₂₀H₄₂	Icosane	282.5	Saturated hydrocarbons	-	-	35.040	1,85
C₂₉H₆₀	Nonacosane	408.8	Saturated hydrocarbons	-	-	37.288	1,89
C₁₉H₄₀	Nonadecane	268.5	Saturated hydrocarbons	-	-	32.261	1,76
C₂₈H₅₈	Octacosane	394.8	Saturated hydrocarbons	40.591	1,93	-	-
C₂₉H₅₀O	Stigmast-5-EN-3-OL-	414.7	Steroids and steroid derivatives	47.196	2,23	47.548	3,54
C₂₉H₅₀O	Stigmast-5-EN-3-OL-	414.7	Steroids and steroid derivatives	-	-	51.078	1,44
C₂₉H₅₀O₂	Vitamin E	430.7	Prenol lipids	41.928	8,09	42.038	4,77
C₂₉H₄₈O	Stigmasterol	412.7	Steroids and steroid derivatives	45.424	2,55	45.672	2,64
C₁₆H₃₀O	Z,Z-11,13-Hexadecadien-1-ol	238.41	Fatty acid derivative	-	-	32.516	1,57

The main compounds in the leaf extract found with a high percent peak area are 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)- or Squalene compounds (39.01%), 2,4',6-Trimethyldiphenylsulfone (14.81%), Vitamin E (8.09%), and 2,6,10-Trimethyl, 14-Ethylene-14-Pentadecne (5.64%), (2E)-3,7,11,15-Tetramethyl-2-Hexadecene-1-OL- (4.17%). There are several compounds with almost the same peak area, namely Stigmasterol (peak area: 2.55%), -6,7,8,9-Tetrahydro-3-Methoxymethyl-5,9--Dimethyl-8-Hydroxy-Naptho- [1,2-B] Furan (2.34%) and two compounds with the same peak Stigmast-5-EN-3-OL and Alpha Amyrin (2.23%).

The compound -6H-Pyrido [4,3-B] -Carbazole-5-Carboxaldehyde, 11-Methyl- belongs to the class Carbazoles and is the main phytoconstituent in the flower H. longiflora (L.) G. Don, indicating the presence of a dominant influence of function on the flower. Other compounds with a reasonably high peak are Vitamin E (4.77%) and hentriacontane (3.87%). The rest of the compound was found with almost the same peak area. 9,12-Octadecadienoic acid (1.08%) was found to be the compound with the lowest biological activity in the extracted compound in H. longiflora (L.) G. Don flowers. The nonane 8-Methyl-7-Thiabicyclo [4.2.1] compound is a new compound that cannot be detected in the database.

From a total of 37 compounds isolated from H. longiflora (L.) G. Don leaf and flower extracts, there are 7 compound variant equations with different percentages and retention times (Table 2). Compounds -2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-,(all-E)-, 2,6,10-Trimethyl, 14-Ethylene-14-Pentadecne, Stigmast-5-EN-3-OL, Stigmasterol, and Vitamin E are bioactive compounds found in flowers as well as leaves. The retention time of compounds -2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-,(all-E)- on the leaves are not much more significant when compared to the same compound in the flower part, and this suggests that the leaves take longer to come out. The compound also has a high percentage of areas in the leaf part. As for the compound 2,6,10-Trimethyl, 14-Ethylene-14-Pentadecne has the same retention time on the leaf part of the flower. The repeated retention time belongs to the compound Stigmast-5-EN-3-OL; in the leaves, this compound has only one-time retention, but in flowers, it has two-time retention. The bioactive constituents of stigmasterol and Vitamin E indicate a stable retention time in the leaves and flowers of H. longiflora (L.) G. Don. The compound -3,7,11,Trimethyl-dodeca-2,4,6,10-Tetraenal was found only on the leaves alone but had a different time interval to exit the column and reach the detector, while 12-Methoxy-18-Nor-5.Beta.-Podocarpa-8,11,13-Trien-3.Alpha. O.L. is only found in the flower section and has a different retention time.

Various potentials are found in the content of leaf extracts and flowers of H. longiflora (L.) G. Don (Table 3). Squalene or by other names 2,6,10,14,18,22-Tetracosahexaen, -2,6,10,15,19,23-hexamethyl-,(all-E)- (peak area: 39,01%) is a natural triterpene hydrocarbon. It is an essential intermediary in intensifying many bioactive secondary metabolites⁵². This compound has various benefits for -human- health, such as anti-cancer effects, cardiovascular disease, and boosting the immune system⁵³. In addition, it also has antibacterial properties ⁵⁴.

In H.longiflora (L.) G. Don flowers, there are bioactive compounds, namely -6H-Pyrido [4,3-B] Carbazole-5-Carboxaldehyde, 11-Methyl- (41.3%) or with the compound name ellipticine. This bioactive constituent is a pyridocarbazole type plant alkaloid that exhibits cytotoxic activity against tumor cells. It is known that ellipticine has exciting photophysical properties in different solvents⁵⁵. It has pharmacological potential as an anti-tumor and anti-neoplastic⁵⁶. Anti-neoplastic agents are drugs that prevent, kill, or inhibit the growth and spread of cancer cells. Most anti-neoplastic agents act on molecular targets important for cell division⁵⁷.

Then it was found that there were eight compounds in flowers and leaves whose biological activity was unknown. Compound -12-Methoxy-18-Nor-5.Beta.-Podocarpa-8,11,13-Trien-3.Alpha.-OL, 8-Methyl-7-Thiabicyclo- [4.2.1] nonan, 9,12-Octadecadienoic Acid, Hexadecane, 2,6,10,14-Tetramethyl-, Nonadecane on flowers has not found its biological activity. Other compounds in the leaves that until now are still unknown in biological activity are 2-[4-(-1 Methylethoxy) bnztl]-4,6-pyrimidinediol, 2-Hydroxy-2-Phenyl-1-Acenapthenon, -6,7,8,9--Tetrahydro-3--Methoxymethyl-5, -9-Dimethyl-8-Hydroxy-Naptho [1,2-B] Furan.

Table 3: Biological activities of H. longiflora (L.) G. Don

Compounds	Part	Biological Activity
Benzaldehyde	Flower	Flavoring agents ⁵⁸ and irritants ⁵⁹
(2E)-3,7,11,15-Tetramethyl-2-Hexadecene-1-OL	Leaf	Antioxidant ⁶⁰, anti-infective and antiparasitic ⁶¹
(9E)-9-Octadecenoic Acid	Leaf	Antifungal ⁶²
12-Methoxy-18-Nor-5.Beta.-Podocarpa-8,11,13-Trien-3.Alpha.-OL	Flower	Not Found
1-Phenylethanone	Leaf	Anti-diabetic and anti-adipogenic ⁶³
2,6,10-Trimethyl, 14-Ethylene-14-Pentadecne	Leaf and Flower	Antioxidant, Antiproliferative, and Anti-cancer ⁶⁴
2,4',6-Trimethyldiphenylsulfone	Leaf	Antibacterial and anti-cancer ⁶⁵
2,6,10,14,18,22-Tetracosahexaen, 2,6,10,15,19,23-hexamethyl-,(all-E)-	Leaf and Flower	Antibacterial ⁵⁴ and insecticide ⁶⁶
2-[4-(-1 Methylethoxy) bnztl]-4,6-pyrimidinediol	Leaf	Not Found
2-Hydroxy-2-Phenyl-1-Acenapthenon	Leaf	Not Found
3,7,11, Trimethyl-dodeca-2,4,6,10-tetraenal	Leaf	Anti-depressants ⁶⁷
4,4,6a,6b,8a,11,12,14b,Octamethyl-1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-Octadehydro-2H-Picen-3-one-	Flower	Anti-cancer ⁶⁸
6,7,8,9-Tetrahydro-3--Methoxymethyl-5,9-Dimethyl-8-Hydroxy-Naptho [1,2-B] Furan	Leaf	Not Found
6H-Pyrido [4,3-B] - Carbazole-5-Carboxaldehyde, 11-Methyl-	Flower	Antitumor dan antineoplastic ⁵⁶
8-Methyl-7-Thiabicyclo [4.2.1] nonan-	Flower	Not Found
9,12-Octadecadienoic Acid	Flower	Not Found
Acteamine, N-(1,1,3,3-tetramethylbutyl)-	Flower	Organic synthesis and chemical medicine ⁶⁹
Acetophenone	Flower	Antifungal ⁷⁰ and flavoring agents ⁷¹
Alpha Amyrin	Leaf	Anti-inflammatory, anxiolytic, and anti-depressant ⁷²
Hentriacontane	Flower	Anti-inflammatory, anti-tumor, and Antibacterial ⁷³
Hexadecane, 2,6,10,14-Tetramethyl-	Flower	Not Found
Icosane	Flower	It has thermal properties ⁷⁴
Nonacosane	Flower	The main paraffin of most plant waxes ⁷⁴
Nonadecane	Flower	Not Found
Octacosane	Leaf	Antioxidant ⁷⁵
Stigmast-5-EN-3-OL	Leaf and Flower	Anti-diabetic, cholesterol reduction and anti-cancer ⁷⁶
Stigmasterol	Leaf and Flower	Immune response, increasing the concentration of enzymes ⁷⁷, and -anti-cancer ⁷⁸
Vitamin E	Leaf and Flower	Chronic disease prevention, antioxidants, and neurological therapy ⁷⁹
Z,Z-11,13-Hexadecadien-1-ol	Flower	Medical, pharmaceutical, and cosmetic components ⁸⁰

CONCLUSION:

This study was one of the few reports in which the bioactive compound extracts of H. longiflora (L.) G. Don were reported. Various bioactive compounds were found in H. longiflora (L.) G. Don. Thirty-seven chemical constituents of H. longiflora (L.) G. Don leaf and flower extracts were identified with GC-MS. Squalene, 2,4',6- Trimethyldiphenylsulfone and Vitamin E which predominate in the leaves act as a producer of high amounts of antioxidants and also as a medium for cancer treatment. Ellipticine, Vitamin E, and hentriacontane predominate in flowers with their role as anti-neoplastic agents that can prevent, kill, or inhibit the growth and spread of cancer cells. Squalene and other bioactive compounds highlight the use of drugs and pharmaceuticals. Based on phytocomponents identified with potential biological activity, this plant can be an essential ingredient in medicine. Its ability as an anti-tumor, anti-cancer, antibacterial, and antioxidant drug from this plant as a natural constituent source can be explored further.

ACKNOWLEDGMENTS:

The author would like to thank the ELSA Botanical Identification Services and Herbarium Bogoriense, National Research and Innovation Agency (BRIN), Indonesia.

COMPETING INTERESTS:

The authors declare that they have no competing interests.

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Received on 26.01.2023 Modified on 18.05.2023

Research J. Pharm. and Tech 2023; 16(11):5347-5353.

DOI: 10.52711/0974-360X.2023.00866