87 urn:lsid:arphahub.com:pub:A116C711-4C18-5A38-8F1E-5E97753A8A64 Folia Medica FM 0204-8043 1314-2143 Plovdiv Medical University 10.3897/folmed.64.e58086 58086 Original Article Experimental pharmacology Phytochemical evaluation of Plumbago zeylanica roots from Indonesia and assessment of its plumbagin concentration Purwoko Mitayani 1 6 mitayani.dr@gmail.com https://orcid.org/0000-0002-3936-3883 Sentono Harijono Kario 2 Purwanto Bambang 3 Indarto Dono 4 5 Department of Medical Biology, Faculty of Medicine, Muhammadiyah University of Palembang, South Sumatera, Indonesia Muhammadiyah University of Palembang Palembang Indonesia Department of Dermato-Venereology, Faculty of Medicine, Dr Moewardi Hospital, Universitas Sebelas Maret Surakarta, Central Java, Indonesia Universitas Sebelas Maret Surakarta Surakarta Indonesia Department of Internal Medicine, Faculty of Medicine, Dr Moewardi Hospital, Universitas Sebelas Maret Surakarta, Central Java, Indonesia Muhammadiyah University of Palembang Palembang Indonesia Department of Physiology, Faculty of Medicine, Universitas Sebelas Maret Surakarta, Central Java, Indonesia Universitas Sebelas Maret Surakarta Surakarta Indonesia Biomedical Laboratory, Universitas Sebelas Maret Surakarta, Central Java, Indonesia Muhammadiyah University of Palembang Palembang Indonesia Faculty of Medicine, Universitas Sebelas Maret Surakarta, Central Java, Indonesia Universitas Sebelas Maret Surakarta Surakarta Indonesia

Corresponding author: Mitayani Purwoko, Department of Medical Biology, Muhammadiyah University of Palembang, Jalan K.H. Balqhi, 13 Ulu, Seberang Ulu I, South Sumatera, Palembang, Indonesia; Email: mitayani.dr@gmail.com; Tel.: +6281320074327

 2022 28 02 2022 64 1 96 102 9233728A-F572-5700-B3C9-670D016966EE 14 09 2020 04 11 2020 Mitayani Purwoko, Harijono Kario Sentono, Bambang Purwanto, Dono Indarto This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Introduction: Plumbago zeylanica grows widely in many tropical countries. In Indonesia, this plant, known as Daun Encok, has some beneficial effects on human health.

Aim: This exploration study aimed to identify the plumbagin compound in P. zeylanica roots from Indonesia.

Materials and methods: Dried roots of P. zeylanica were manually ground and then the powder was macerated using ethanol and chloroform for 24 hours at room temperature. All extracts of P. zeylanica were then analyzed using gas chromatography-mass spectrometry (GC-MS). Plumbagin concentration was measured by comparing the extract with pure plumbagin.

Results: GC-MS analysis of ethanol extract and chloroform extract of P. zeylanica roots showed the presence of plumbagin as the highest peak. Plumbagin concentration in ethanol extract was 13%, while in chloroform extract it was 81%.

Conclusions: The chloroform extract of P. zeylanica root from Indonesia demonstrates a higher concentration of plumbagin compared to ethanol extract.

 chloroform root extract GC-MS medicinal plant Citation

Purwoko M, Sentono HK, Purwanto B, Indarto D. Phytochemical evaluation of Plumbago zeylanica roots from Indonesia and assessment of its plumbagin concentration. Folia Med (Plovdiv) 2022;64(1):96-102. doi: 10.3897/folmed.64.e58086.

 Introduction

Plumbago zeylanica, which is commonly known as Daun Encok or Ki Encok in Indonesia, belongs to the family Plumbaginaceae. This plant grows in many countries and is recognized by the community as having many benefits on human health; for example, in treating joint pain and skin diseases.[1] Several previous studies have found other benefits of this plant, namely as a nephroprotective agent, antiproliferation of cancer cells, anti-parkinsonism agent, antibacterial agent, analgesic, and anti-inflammation.[2–7] However, only a few of those studies were conducted using Plumbago zeylanica from Indonesia.

The best known bioactive marker in P. zeylanica is plumbagin. Plumbagin is effective as an antiproliferative, antimalarial, and antibacterial agent.[3,8,9] In a previous study, GC-MS was used to find the compound contained in ethanol extract and diethyl extract of P. zeylanica root, but they did not find plumbagin.[2,10] Plumbagin concentration in ethanolic extract of root part has been found to be low (1.9%).[11] There is no publications about plumbagin contained in P. zeylanica plants from Indonesia.

 Aim

This study aimed to identify the plumbagin compound contained in the root part of P. zeylanica plants from Indonesia.

 Materials and methods Plantation of <italic><tp:taxon-name><tp:taxon-name-part taxon-name-part-type="genus" reg="Plumbago">P.</tp:taxon-name-part> <tp:taxon-name-part taxon-name-part-type="species" reg="zeylanica">zeylanica</tp:taxon-name-part></tp:taxon-name></italic>

The P. zeylanica seeds were obtained from Akhyar Flora, Bekasi, West Java, Indonesia and then were grown in pots 25-40 cm in diameter filled with soil. In order to stimulate seed sprout, it was watered twice a day with tap water and was supplemented with a commercial fertilizer containing 16% nitrogen (6.5% nitrate-N and 9.5% ammonium-N), 16% phosphate (P2O5), and 16% potassium (K2O). After 7 months, grown P. zeylanica plants had some and big enough roots that could be used for the next extraction process. The P. zeylanica plant was authenticated by a botanist, Mrs. Susi Dewiyeti, S.Si, M.Si, which was deposited at the Department of Biology, Faculty of Education, Universitas Muhammadiyah Palembang, South Sumatera, Indonesia.

P. zeylanica roots were cut from their plants in the afternoon and washed thoroughly under tap water to remove soils and other debris. Cleaned roots were air-dried for 18 days in room temperature by exposing them to air flow and indirect sunlight. The average of relative humidity in Palembang remained constant (94.9% RH) during those days and the average of sun rays was 5.6 hours per day. Finally, air-dried roots were mechanically ground to make powder using a blender.

 Extraction of <italic><tp:taxon-name><tp:taxon-name-part taxon-name-part-type="genus" reg="Plumbago">P.</tp:taxon-name-part> <tp:taxon-name-part taxon-name-part-type="species" reg="zeylanica">zeylanica</tp:taxon-name-part></tp:taxon-name></italic> roots

A total of 10 g of root powder was soaked in either 100 ml ethanol (Emsure®, Merck, Germany) or 100 ml chloroform (Emsure®, Merck, Germany) for 24 hours. These root powder solutions were filtered with filter paper and the filtered solutions were concentrated using a rotary evaporator (B-One Rotary Evaporator Model RE-1000VN) with 52 rpm at 60°C for 30 minutes for the ethanol extract and 10 minutes for the chloroform extract. The extract yield of 100 g roots powder with ethanol solvent was 4.6% whilst 1.7% was for chloroform solvent. All crude extracts were finally stored in the refrigerator at 4°C until further analysis.

 Chemical analysis of <italic><tp:taxon-name><tp:taxon-name-part taxon-name-part-type="genus" reg="Plumbago">P.</tp:taxon-name-part> <tp:taxon-name-part taxon-name-part-type="species" reg="zeylanica">zeylanica</tp:taxon-name-part></tp:taxon-name></italic> extracts using gas chromatography-mass spectrometry

The ethanol and chloroform extracts of P. zeylanica roots was analysed using Thermo Scientific Trace 1310 Series (Thermo Fisher Scientific, San José, CA, USA) with a Trace ISQ LT mass detector (San José, CA, USA) (GC-MS) and a Thermo Scientific AI 1310 automatic injector (San José, CA, USA). The chemical analysis was conducted in a ZB-5MS (30 m × 0.25 mm × 0.25 µm) column (Phenomenex®, Torrance, CA, USA) and the injector temperature was set at 300°C. The injection mode was splitless and the injection volume was 1 μL. The plumbagin compound was identified and matched with the peak of main EI MS Library (mainlib) from NIST/EPA/NIH. Chemical structure of all components was made by Chem Draw software (PerkinElmer, US).

 Results

The raw extracts of P. zeylanica roots were examined by GC-MS. GC-MS examination results for the two samples are shown in Fig. 1 below. GC-MS for each extract showed 50 peaks and all peaks are known. But, in this article, only 10 highest % area were shown in Tables 1, 2.

Details on the compound of each extract are shown in Tables 1, 2 and 3.

Based on Table 1 and Table 2, there were 2 similar compounds: 5-hydroxy-2-methyl-1,4-naphthalenedione (plumbagin) and 2-Allyl-1,4-dimethoxy-3-methyl-benzene.

Because of the highest GC-MS curves for both extracts showed 5-hydroxy-2-methyl-1,4-naphthalenedione (or plumbagin), we then confirmed the presence of plumbagin using pure plumbagin (Sigma Aldrich, lot #SLBZ1960). The purchased plumbagin as an internal control contains 5-hydroxy-2-methyl-1,4-naphthalenedione. The final concentration of plumbagin in chloroform solvent was about four times higher than the ethanol solvent (Table 4) based on the formula below (Fig. 2).

DC66C94A-5252-51D3-BF40-E52A13A90AF9

GC-MS examination results. (a) The roots of P. zeylanica macerated using absolute ethanol; (b) The roots of P. zeylanica macerated using chloroform (CHCl3).

https://binary.pensoft.net/fig/674937 A536B253-4889-5441-8255-E814E8E0B386

The curve of plumbagin concentration measured by GC-MS.

https://binary.pensoft.net/fig/674938

Chemical compounds in ethanol extract of P. zeylanica roots (sorted by low to high RT)

No. Compound name MF MW RT % area
1 Dihydroxyacetone C3H6O3 90 4.57 1.47
2 Glycerin C3H8O3 92 8.89 1.02
3 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O4 144 8.98 1.39
4 5-Hydroxymethylfurfural C6H6O3 126 10.00 1.13
5 Sucrose C12H22O11 342 12.07 7.07
6 5-hydroxy-2-methyl-1,4-naphthalenedione C11H8O3 188 13.60 56.88
7 2-Allyl-1,4-dimethoxy-3-methylbenzene C12H16O2 192 14.42 9.59
8 11-decyltetracosane C34H70 478 19.64 0.99
9 11-decyltetracosane C34H70 478 24.52 3.26
10 Cyclodecasiloxane, eicosamethyl- C20H60O10Si10 740 25.61 1.01

MF: molecular formula; MW: molecular weight; RT: retention time

Chemical compounds in chloroform extract of P. zeylanica roots (sorted by low to high RT)

No. Compound name MF MW RT (min) % area
1 5-hydroxy-2-methyl-1,4-naphthalenedione C11H8O3 188 13.64 71.97
2 2-Allyl-1,4-dimethoxy-3-methyl-benzene C12H16O2 192 14.44 11.15
3 Eicosane C20H42 282 18.35 0.67
4 Heneicosane C21H44 296 19.65 0.68
5 Eicosane C20H42 282 20.82 0.87
6 Eicosane C20H42 282 21.88 0.97
7 Eicosane C20H42 282 22.86 0.99
8 Eicosane C20H42 282 23.77 1.20
9 Tetratriacontane C34H70 478 24.65 1.46
10 Tetratriacontane C34H70 478 25.62 1.55

MF: molecular formula; MW: molecular weight; RT: retention time

Bioactive compounds identified using ethanol and chloroform extracts of P. zeylanica roots

No Compound name Structure Activity on Human Health
1 5-hydroxy-2-methyl-1,4-naphthalenedione - Antiproliferative[3] - Antimalarial[8] - Antibacterial[9]
2 Dihydroxyacetone - Skin tanning agent[12]
3 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- Antioxidant[13]
4 5-Hydroxymethylfurfural - Antioxidant[14] - Anti-allergy[14] - Anti-inflammation[14] - Antihypoxia[14] - Antihyperuricemia[14]
5 Cyclodecasiloxane, eicosamethyl- - Antimicrobial[15] - Antihelmintic[15] - Antioxidant[15] - Hepatoprotective[15]
6 Sucrose Hyperlipidemic agent[16]
7 Glycerin Skin hydrating agent[17]
8 Heneicosane Anticancer[18]
9 Tetratriacontane Anticancer[18]
10 11-decyltetracosane Unknown
11 2-Allyl-1,4-dimethoxy-3-methyl-benzene Unknown
12 Eicosane Unknown
Discussion

Plumbagin is known as an anticancer agent. Plumbagin demonstrates the activation of autophagy, apoptosis, and cell cycle arrest in some cancers. Plumbagin also exhibits the capability of angiogenesis inhibition in some cancers.‌[19] The selection of chloroform as solvents in this study was based on the results of the previous study. Plumbagin is naphthoquinone. Quinone in Plumbago zeylanica roots only detected in extract dissolved with chloroform.‌[2] Chloroform or trichloromethane belongs to the Class 2 solvent that is non-genotoxic carcinogens for the animal. Chloroform has a possible toxicity that is reversible.[20]

A previous study mentioned that the concentration of plumbagin in ethanol extract of Plumbago zeylanica root was only 1.9%.[11] In this study, we aimed to identify the plumbagin content in the roots of Plumbago zeylanica from Indonesia. We found a higher concentration of plumbagin that was extracted using chloroform. A high concentration of plumbagin in chloroform extract is never been published before, but the amount of 81% concentration in this study (Table 4) is similar to plumbagin concentration in petroleum ether extract of P. zeylanica in a previous study.[21]

This study found 56.88% area of plumbagin in GC-MS result of ethanolic extract (Table 1). This area is higher than the previous study which only found plumbagin in 7.94% area of ethanolic extract.[22] Some previous study used cultivated plants from India, while this study used cultivated plants from Indonesia. The geographical location of cultivation may give different results of the phytochemical contents.

The concentration of plumbagin in each sample

Solvent type Sample weight Sample volume Concentration
(g) (ml) % ppm
Chloroform 0.0079 5 81 805340
Ethanol 0.0102 5 13 128718
Conclusions

It can be inferred from the current study that plumbagin concentration is four times higher in chloroform extract compared to ethanol extract as revealed through the GC-MS technique. Hence, chloroform solvent is suggested to be used to exploit the potential of plumbagin from P. zeylanica as herbal medicine.

 Acknowledgments

Authors are thankful to the Integrated Laboratory of Chemistry Study Program, Universitas Sriwijaya, Palembang, Indonesia for providing GC-MS examination. Authors are thankful to Biomedical Laboratory of Faculty of Medicine, Universitas Muhammadiyah Palembang, Indonesia for plant extraction service. Authors are thankful to Indonesia Endowment Fund for Education (LPDP), Ministry of Finance, Republic of Indonesia for the scholarship. Authors are thankful to I Gede Alit Pujawan, Muhammad Ainun Najib, and Muhammad Fadhil Rizki Martha for the chemical drawing.

Conflict of Interest

The authors declare no conflict of interest.

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