Arabian Journal of Chemistry (2020) 13, 8888–8897

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

New potential and characterization of Andrographis

paniculata L. Ness plant extracts as photoprotective
agent
Qonitah Fardiyah a,b, Taslim Ersam c, Suyanta d, Agus Slamet e, Suprapto a,
Fredy Kurniawan a,*

a
Instrumentation and Analytical Sciences Laboratory, Department of Chemistry, Faculty of Sciences, Institut Teknologi Sepuluh
Nopember, Surabaya 60111, Indonesia
b
Analytical Chemistry Laboratory, Departement of Chemistry, Faculty of Mathematic and Natural Science, Brawijaya
University, Malang 65145, Indonesia
c
Natural Products and Synthesis Chemistry Research Laboratory, Department of Chemistry, Faculty of Sciences, Institut
Teknologi Sepuluh Nopember, Surabaya 60111, Indonesia
d
Analytical Chemistry Laboratory, Department of Chemistry Education, Faculty of Mathematics and Natural Science,
Yogyakarta State University, Indonesia
e
Department of Environmental Engineering, Faculty of Civil, Planning, and Geo Engineering, Institut Teknologi Sepuluh
Nopember, Surabaya 6011, Indonesia

Received 10 August 2020; accepted 6 October 2020

Available online 17 October 2020

KEYWORDS Abstract The species Andrographis paniculata L. Ness, family Acanthaceae grows in many parts in
Herbal plant; the island of Java in Indonesia. In this research, new potentials of photoprotective agents from A.
Sambiloto; paniculata plant extracts, along with their characteristics, were investigated. The phytochemical test,
Ethanolic extracts; infrared analysis, ultraviolet characterization, fluorescence identification, flavonoid content by thin
UV protector; layer chromatography, high performance liquid chromatography-mass spectrometry (LCMS/MS)
SPF and photoprotective analysis were performed in the extracts. The new potentials of A. paniculata
plant extracts were studied at various concentrations of extracts and shown by the value of the
sun protection factor (SPF). Based on phytochemical test, A. paniculata plant extracts contained
flavonoids, alkaloids, tannins, triterpenoids and polyphenols. A. paniculata plant extracts have 2
maximum wavelengths at 230 nm and 362 nm. The excitation peak at 300 nm (552.11 a.u) and
the emission peak at 605 nm (516.02 a.u). The flavonoid content of the extracts was 0.022 mg/mL
quercetin with Rf value of 0.61. The chromatogram of the LCMS/MS is similar to quercetin-3-

* Corresponding author.
E-mail address: [email protected] (F. Kurniawan).
Peer review under responsibility of King Saud University.

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New potential and characterization of Andrographis paniculata L. Ness plant extracts as photoprotective agent 8889

glycoside standard at a retention time of 2.77. The photoprotective activity of A. paniculata plant
extracts had
15 SPF. Its shows the possibility to use this extract as photoprotective agent in phar-
maceutical preparations.
Ó 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open
access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction The content of organic compounds in A. paniculata plants can be used

as a natural active agent in sunscreen formulations. The performance
Andrographis paniculata L. Ness (sambiloto) plant thrives and is com- of this active agent depends on the capacity of the active ingredient
monly found in Indonesia, in particular on the island of Java. A. pan- in absorbing ultraviolet radiation. The effectiveness of the active
iculata is an important medicinal plant species which belong to the agents is measured by its absorption value at ultraviolet wavelengths
Acanthaceae family which grows wild in the open. Sambiloto leaves and is expressed as the value of the sun protection factor (SPF)
have a physical appearance similar to a green colour and appear bright (Dutra et al., 2004; Olayemi et al., 2017; Patil et al., 2015). According
in the sun. A. paniculata plants have been widely used to solve various to the Food and Drug Administration of the United States and Euro-
health problems. Medicinal plants are important sources of valuable pean Union (FDA), the recommended value of sun protection factor is
therapeutic agents, both in traditional and modern medicine. When SPF-15 or higher so that it is able to reduce the risk of skin cancer and
compared to other medicinal plants, A. paniculata is one of the most premature aging (Gabros et al., 2020). Thus, active agents that can
potential herbs to be used as an alternative treatment for treating var- provide high SPF values and increase filtering efficiency against ultra-
ious deadly diseases. This is due to A. paniculata plant extracts have violet radiation exposure are needed.
antihypertensive effect and have the ability of reducing the conversion A. paniculata plant extracts which are rich in organic compounds
of plasma angiotensin into enzyme (ACE) and lipid peroxidation in have the potential to have high SPF values and are expected to provide
kidney (Akbar, 2011). In addition, this plant contains antimicrobial effective protection against ultraviolet radiation exposure. The pres-
activity (Singha et al., 2003), antioxidants and anti-inflammatory ence of flavonoid and phenolic compounds in A. paniculata plant
(Chandrasekaran et al., 2010), antihyperglycemic and hypoglycemic extracts causes it to have photoprotective ability against ultraviolet
activity (Zhang and Tan, 2000). The abilities of antioxidant, antimicro- radiation. However, research on this subject has never been taken fur-
bial, anticancer and antimalarial activities of A. paniculata plants have ther in previous studies. On this basis, new potentials of A. paniculata
been previously researched by Chuah et al. (2017), Kumar et al. (2004), plant extracts as a protector for ultraviolet radiation and its character-
Mishra et al. (2009). istics were developed and analyzed using various methods. The results
Based on the results of the previous studies (Ismail et al., 2017; show some possibilities to use of A. paniculata plant extracts as a pho-
Jarukamjom and Nemoto, 2008; Praveen et al., 2014), it is known toprotective agent in pharmaceutical preparations.
that A. paniculata plants contain flavonoid and phenolic compounds.
Flavonoid compound is one of the organic compounds found in
many types of plants. This compound helps biological processes that 2. Experimental work
occur in plants, in particular processes that involve antimicrobial
activity (Kurniawan et al., 2014), antioxidants and photoprotective. 2.1. Reagents solutions and instruments
Photoprotective activity is related to radiation emitted by sunlight,
known as ultraviolet radiation. Continuous ultraviolet radiation
Andrographis paniculata L. Ness plant material obtained from
exposure can have positive and negative effects on human life
(Cooley and Quale, 2013; Jain and Jain, 2010). The negative effects Materia Medica Batu Technical Implementation Unit, Depart-
of ultraviolet radiation include photoaging and photocarcinogenic. ment of Health, East Java Province of Indonesia, with the key
Photoaging can cause sagging and wrinkling of the skin, whilst pho- to plant determination1b-2b-3b-4b-6b-7b-9b-10b-11b-12b-13b
tocarcinogenic causes damage to cells and DNA. Continuous sun -14b-16a-239b-244b-248b-249b-250a-251b-253b-254b-255a-25
exposure can be a risk factor for nonmelanoma skin cancer 6a-257b-259a-2b. Ethanol 96% E.Merck, reagent for phyto-
(Moloney et al., 2002). Various chromophores in the skin, such as chemical tests, all chemicals used in this study have a high
melanin, DNA, RNA, protein, amino acids and others can absorb degree of purity (pro analyst). All glassware’s used in this
ultraviolet radiation. DNA is an important macro molecule that experiment were washed and rinsed with demineralised water
can mutate and cause malignant transformation of skin cancer cells
before being used. Instrumentation used for characterization
(Narayanan et al., 2010). Photochemical reactions due to the absorp-
included FTIR spectrophotometer (Agilent 5500 FTIR) used
tion of ultraviolet radiation by chromophores involve reactive oxygen
species (ROS). This species has a dangerous effect if ultraviolet to determine the specific wavelength of A. paniculata plant
radiation is emitted continuously (Balogh et al., 2011; Diffey, extracts through functional group detection, UV–Vis spec-
2007). trophotometer (Thermo Sciencetific-Genesys 10 s UV Visible)
The use of UV filter types chemical sunscreen generally involves to determine the maximum wavelength of A. paniculata plant
organic compounds which works by absorbing ultraviolet radiation extracts and to analyze the SPF value data from A. paniculata
(Salvador and Chisvert, 2007). Natural organic compounds are found plant extracts. Other instruments used include, Fluorescence
in plant extracts. The need for plant extracts which contain a lot of fla- spectrophotometer (Perkin Elmer LS 55 fluorescence spec-
vonoid compounds become an important component in the discovery trometer connected with FL Winlab software) to determine
of new active molecules for human photoprotective (Costa et al., 2015;
the wavelength of excitation and specific emissions from A.
Lin et al., 2016). Plant extracts that are rich in flavonoids are able to
paniculata plant extracts, High Performance Thin Layer Chro-
absorb ultraviolet radiation in two regions of maximum wavelength.
Hence in general, they generate two maximum peaks of absorption matography (HPTLC) to determine the flavonoid as querce-
of ultraviolet radiation in the regions of UV A (320–400 nm) and tine and Liquid Chromatography Mass Spectrometry/Mass
UV-B (290–320 nm) (Baron and Suggs, 2014; Costa et al., 2015). Spectrometry (LCMS/MS).
8890 Q. Fardiyah et al.

2.2. Preparation of plant extracts wavelengths of 200 nm to 400 nm (Napagoda et al., 2016).
The wavelengths with maximum absorbance peaks are
The fresh leaves of A. paniculata was washed using aquadest, recorded as maximum wavelengths of A. paniculata plant
then dried in the open air for 5 days while protected from extracts. The maximum absorbance peak in the spectra
direct sunlight. The dry leaves were crushed using a grinder. obtained shows the specific character of A. paniculata plant
The obtained powder called simplicia was stored in a dry extracts.
tightly closed container. This container was stored at room
temperature before used (Napagoda et al., 2016; Patil et al., 2.6. Fluorescence identification
2015). 250 g of simplicia powder from the leaves of A. panicu-
lata plant was weighed using the analytical balance of the A. paniculata plant extracts were identified by excitation wave-
Ohauss brand and extracted using an ethanol 96% E Merck length and emission using the Perkin Elmer LS 55 fluorescence
E solvent in a ratio of 1:10. The extraction process used a mac- spectrophotometer. Prior to measurement, fluorescence instru-
eration method for 72 h. Maceration results are filtered using mentation was adjusted at scan speed of = 500, SlitEx/Em =
Whatman filter paper No. 41 with a vacuum pump. The filtrate 10 nm/10 nm. The initial step taken is to determine the wave-
obtained was then concentrated with a vacuum rotary evapo- length at maximum intensity using a fluorescent spectrometer.
rator at 50 °C until a thick extract was obtained. The obtained Subsequently, pre-scan the sample at kex = 200–800, kem =
extracts was kept at 4 °C in the refrigerator before used 200–900 nm where these are then noted as excitation wave-
(Mishra et al., 2012; Thibane et al., 2019) length and emission wavelength at maximum intensity. The
sample is measured at the wavelength that has been obtained
2.3. Phytochemical test previously. Sample measurements were carried out 5 times.
The excitation and the emission monochromator slits were
Phytochemical tests were carried out to determine the presence set to 10 nm. The FL WinLab software was used to register
of flavonoid compounds, polyphenols, alkaloids, terpenoids, the fluorescent signals (Casale et al., 2018).
tannins and saponins in A. paniculata plant extracts. In princi-
ple, the procedure is performed by dissolving 2 mL of A. pan- 2.7. Flavonoids content
iculata plant extracts in 8 mL demineralised water. The
solution is subsequently filtered, and the filtrate is put into sev- A total of 5 mL of A. paniculata plant extracts was injected into
eral test tubes. Next, a few drops of different reagents were the Camag Linomat HPTLC column 5. The stationary phase
added in accordance into each of the test tubes, where changes used was GF254 silica gel with a mobile phase which was a
that occurred were consequently observed. The reagents used mixture of chloroform, formic acid, and methanol mixed at
for the flavonoid test were concentrated HCl and Mg powder. a ratio of 3.3: 0.8: 0.2 respectively (Kshirsagar et al., 2008).
Whereas the FeCl3 reagent was used for polyphenol test and This method is validated by an external calibration curve using
tannin test, Meyer reagent and Dragendroff reagent were used a standard solution of quercetin, prepared in ethanol with five
for alkaloid test, and Bouchardate reagent was used for alka- different concentrations.
loid test and terpenoid test. whilst the hot demineralised water
was used for saponin test (Yadav and Agarwala, 2011). 2.8. LCMS/MS quantification

2.4. Infrared analysis A total of 1 mL of A. paniculata plant extracts was injected

into the LCMS/MS column. The effluent used consisted of 2
Previously, the sampling device (DialPath) was opened by types of solvent A (0.1% formic acid in water) and solvent B
rotating the arm and the bottom window had to be seen. Dro- (0.1% formic acid in acetonitrile). The column used was the
plets of a small sample of A. paniculata plant extracts were put Phenomenex type. This method is validated by an external cal-
in the sample window located on the DialPath base plate. The ibration curve using a standard solution of quercetin, prepared
sample window is a 2 mm diameter yellow material held by a in ethanol with four different concentrations. The standard
metal disk around it. The sample is confirmed to have covered quercetin-3-glycoside solution is injected, and line graphs are
the entire surface area of the lower window. A. paniculata plant obtained on Microsoft Office Excel Ó by calculating the aver-
extracts are ready for infrared analysis with a transportable age. The accuracy is expressed as a similarity between the value
diamond crystal attenuated total reflectance-Fourier transform measured experimentally and the reference value specified. The
infrared (ATR-FTIR) spectrometer (Agilent Technologies values of accuracy are calculated according to the formula
5500a). This instrument was used to collect spectra in the RSD (%) = (SD  100)/C, where RSD (%) is precision, SD
650–4000 cm1 region. No sample preparation was required is the standard deviation and C is the average concentration
directly analysed on the diamond window. Intimate contact calculated. As for the accuracy, it is calculated by using Accu-
of the sample with the crystal window was required to give racy (%) = (Cexp  100)/TC, where Cexp is the total querce-
good quality spectra (Mitchell et al., 2013). tin concentration of the extract and TC is the theoretical
standard reference concentration. The detection limit (LOD)
2.5. UV characterization and the quantification limit (LOQ) are estimated by the slope
whereas the mean and the standard deviation of the concentra-
A. paniculata plant extracts are dissolved in a particular vol- tion are used to construct the analytical curve (Costa et al.,
ume of ethanol solvent. The solution is measured at 2015; Santhanam et al., 2017)
New potential and characterization of Andrographis paniculata L. Ness plant extracts as photoprotective agent 8891

2.9. Preparation of photoprotective analysis macokinetic behavior of the constituents present in herbal
preparations (Mehta et al., 2017).
A. paniculata plant extract was dissolved in 100 mL of ethanol
solvent. The solution was ultrasonified for 5 min, and subse- 3.1. Phytochemical test
quently filtered by rejecting 10 mL of the first filtrate out so
that the first solution was obtained. 10 mL aliquots from the From the phytochemical test results of the A. paniculata plant
first solution are dissolved in 100 mL ethanol and a second extracts, it contains secondary metabolite compounds includ-
solution is obtained. 10 mL aliquots from the second solution ing flavonoids, alkaloids, triterpenoids, tannins and polyphe-
are dissolved in 50 mL ethanol. The sample solution is ready to nols, all of which are listed in Table 1. The compound is a
be measured using an ultraviolet spectrophotometer. Measure- plant defence chemical compound produced in plant tissues
ments were made at wavelengths of 290 nm to 320 nm with (Hsieh et al., 2016; Okhuarobo et al., 2014; Rajagopal et al.,
ethanol as a blank solution. The absorbance readings were per- 2003; Sudhakaran, 2012). The presence of flavonoids is known
formed in triplicate (Patil et al., 2015). to inhibit tumor growth and protect against gastrointestinal
infections. It is also pharmacognostically important to provide
2.10. Sun protection factor (SPF) from extracts evidence for the plants to be utilized in the field of eth-
nomedicine. Some of these bioactive compounds are formed
Photoprotective analysis of A. paniculata plant extracts was as secondary metabolites along with the growth of the plants,
performed by calculating the value of the sun protection factor which also function as antimicrobials to protect plants against
or SPF. SPF value is obtained by calculating the absorption of microbial invasion. Alkaloids have been well researched and
ultraviolet radiation in the wavelength region of UV B (290– developed for several pharmacological properties, including
320 nm) using the Mansur equation. These equations can be antiprotozoal, cytotoxic, antidiabetic properties (Zhang and
written as follows: Tan, 2000), as well as anti-inflammatory properties (Appiah
et al., 2017). The presence of alkaloids is crucial as some
X
320
amounts of it can be used as antimalarial, analgesic and stim-
SPF ¼ CFx ðEEðkÞxIðkÞxAbsðkÞÞ
k¼290
ulant (Churiyah et al., 2015). Tannin compounds in A. panic-
ulata plant extracts are reported to be able to selectively
The SPF value is calculated by applying the Mansur equa- inhibit HIV replication in addition to being used as a diuretic
tion where CF is a correction factor of 10; EE (k) is the erythe- (Hossain et al., 2014). In addition, the content of phenolic
mal efficiency spectrum; I (k) is the spectrum of the sun’s compounds, alkaloids and flavonoids in A. paniculta plant
intensity; and abs (k) is the absorbance of the solution extracts is water-soluble anti-oxidants which functions as an
(Costa et al., 2015; Patil et al., 2015). The value of EE anti-free radical that prevents oxidative cell damage (Sheeja
(k)  I (k) is a constant. A. paniculata plant extracts were dis- et al., 2006). Other advantageous properties of A. paniculta
solved in ethanol to a final concentration of 4, 10, 12, 16, plant extracts are they have a strong anticancer (Kumar
20 mL/mL. The SPF model used in this study is in accordance et al., 2004) and anti-rheumatic activity (Hidalgo et al., 2013).
with the methodology described by Mansur et al. (1986). Phytocompounds are effective in photoprotective activity
Absorbance samples were measured in the UV-B wavelength because they contain phytoconstituents of natural active ingre-
range of 290–320 nm, with an increase of 5 nm and three deter- dients as a substitute for chemicals in photoprotection prepa-
minations were made at each point. rations. Phytocompounds are quite effective in their
photoprotective action against solar radiation. Understanding
2.11. Statistical analysis the pharmacokinetic profile of phytocompounds is very

SPF data were calculated as mean ± SD for five independent

measurements (n = 5). Microsoft Excel 265 was used to per-
form the statistical analysis using one-way ANOVA with Table 1 Phytochemical test result of A. paniculata plant
alpha = 0.05 followed by Least Significant Difference (LSD) extracts.
test. Means were significantly different when P values < 0.05 Compounds Parameter Result
for multiple range and compared each other with LSD test. identification
Flavonoid Brick red, pink, dark red +
3. Results and discussion Alkaloid
Meyer White deposits +
In this work, the selection of ethanol as an organic solvent in Dragendrof Orange deposits –
the maceration process is based on the results of previous Bouchardhat Chocolate deposits –
study conducted by Fardiyah et al. (2018). The results showed Tannin Dark green, dark blue, dark +
brown
that in the maceration process of A. paniculata plants using 3
Terpenoid
types of solvent. The obtained result showed that ethanol
Steroid A mix of blue and green –
has optimum fluorescence intensity value in comparation with Triterpenoid A mix of orange, brown, and +
2 others solvents (ethyl acetate and n-hexane). Different types orange
of solvent extraction will result in difference pharmacokinetic Polifenol Dark green, dark blue, dark +
profile of the bioactive components present in the A. paniculata brown
plant extracts. The type, method of preparation and composi- Saponin Permanent foam –
tion of prescribed herbal preparation can influence the phar-
8892 Q. Fardiyah et al.

important. These phytochemicals compounds show a more 0.952 A (see Table 2). The presence of these two maximum
complex pharmacokinetic profile such as the study of time absorption peaks indicated that A. paniculata plant extracts
course of phytochemicals, absorption, distribution, metabo- has a rich content of flavonoids. This matches with the theory
lism, and excretion (Mehta et al., 2015). Most of the phyto- which states that plant extracts that are rich in flavonoids are
chemical like flavonoids, alkaloids, and terpenoids contain efficient in absorbing ultraviolet rays, typically indicated by
various subclasses and hence it is very essential to know speci- two maximum peaks of ultraviolet absorption. In this case,
fic bioavailability and metabolites associated with each sub- the two maximum peaks occurred between 230 and 280 nm
class. The complexity of the chemical content in plant and the other 300–550 nm (Costa et al., 2015).
extracts is a major challenge (Mehta and Dhapte, 2016).
In this study, the photoprotective mechanism of phytocon- 3.4. Fluorescence identification
stituents in A. paniculata plant extracts was carried out by
absorbing photon energy from the sun at ultraviolet wave- Fluorescence identification of A. paniculata plant extracts pro-
lengths (areas that are harmful to the skin) and then releasing duced two spectra. The obtained spectra are excitation spectra
that energy at visible light wavelengths, which are safe areas shown in Fig. 3 and emission spectra shown in Fig. 4. The
for skin protection. The presence of phytoconstituents in the ultraviolet radiation absorption by A. paniculata plant extracts
ethanol extract of A. paniculata includes flavonoids, alkaloids, was shown at an excitation wavelength of 300 nm with an
tannins, triterpenoids and polyphenols. The existence of these absorption intensity of 552.11, whilst the emission of ultravio-
phytocompounds can increase the ability of the ethanol extract let radiation by A. paniculata plant extracts occurred at an
of A. paniculata to absorb photon energy so that it is quite emission wavelength of 605 nm with an emission intensity of
effective to be used as a photoprotective agent. 516.02. The ability of absorption intensity of A. paniculata
plant extracts was 36.09. In Fig. 3, it is shown that A. panicu-
3.2. Infrared spectra identification lata plant extracts absorb ultraviolet radiation in the UV B
region of 290–320 nm. The presence of flavonoid and phenolic
Identification results of infrared spectra in A. paniculata plant compounds in A. paniculata plant extracts causes this extract
extracts are shown in Fig. 1. Based on Fig. 1, it shows the pres- to absorb ultraviolet radiation in the UV B region. In Fig. 4,
ence of –OH group from the phenolic group at 3294.96 cm1 it shown that the emission of ultraviolet radiation by A. panic-
and 905.74 cm1. Phenolic compounds are compounds that ulata plant extracts occurred in visible areas of 400–800 nm.
are found in plants and have a unique structure that has one This indicates that the A. paniculata plant extracts can be used
or more hydroxyl groups attached to one or more aromatic as a photoprotective agent with its potential to emit ultraviolet
benzene rings (Lin et al., 2016). C-H bonds from alkanes radiation in a safe area, which is the visible area.
appear at 2847.68 cm1 and 2929.69 cm1. A strain between
C‚O from carbonyl group or carboxyl group was also seen 3.5. Flavonoid content
at 1640.03 cm1. There is a –CH3 group at 1438.75 cm1
and a -C-O-C- group at 1203.93 cm1. The existence of a dou- The presence of flavonoid content in A. paniculata plant
ble bond in the gene alkene from an aromatic compound is extracts was analyzed using high performance thin layer chro-
shown at 1032.47 cm1. matography (HPTLC) with a standard solution of quercetin.
From the research results obtained, the HPTLC chro-
3.3. UV characterization matogram of A. paniculata plant extracts showed a Retention
factor (Rf) value of 0.61 (Fig. 5). The value of Rf is defined as
Fig. 2 displays the ultraviolet characterization spectra of A. the ratio of the distance traveled by a compound on the surface
paniculata plant extracts. In Fig. 2, two maximum absorption of the stationary phase divided by the distance traveled by the
peaks are shown. The first peak is seen at wavelength of solvent as the mobile phase. The greater the Rf value of the
230 nm with an absorbance of 0.434 A, whereas the second sample, the greater the movement distance of these compounds
peak is seen at wavelength of 362 nm with an absorbance of on thin layer chromatography (TLC) plates. There are specific

Fig. 1 The infrared spectra of A. paniculata plant extracts.

New potential and characterization of Andrographis paniculata L. Ness plant extracts as photoprotective agent 8893

Fig. 2 Ultraviolet spectra of A. paniculata plant extracts with 2 maximum wavelengths; 230 nm (0.434 A) and 362 nm (0.952 A).

Table 2 Maximum wavelength of A. paniculata plant extracts.

k (nm) A1 A2 A3 Average
200 0.245 0.236 0.252 0.244
210 0.295 0.308 0.319 0.307
220 0.330 0.344 0.322 0.332
225 0.341 0.352 0.354 0.349
230 0.434 0.432 0.435 0.434
235 0.368 0.362 0.368 0.366
240 0.339 0.347 0.338 0.341
250 0.329 0.316 0.328 0.324
260 0.296 0.304 0.287 0.295 Fig. 3 The excitation spectra of A. paniculata plant extracts at
270 0.247 0.253 0.252 0.251 kexcitation 300 nm.
280 0.037 0.036 0.038 0.037
290 0.182 0.182 0.183 0.183
295 0.444 0.444 0.444 0.444
300 0.616 0.615 0.615 0.615
305 0.713 0.713 0.713 0.713
310 0.766 0.766 0.765 0.765
315 0.800 0.799 0.800 0.799
320 0.827 0.826 0.827 0.827
330 0.872 0.874 0.873 0.873
340 0.897 0.896 0.897 0.897
350 0.924 0.925 0.925 0.925
355 0.940 0.939 0.939 0.939
356 0.942 0.941 0.943 0.942
Fig. 4 . The emission spectra of A. paniculata plant extracts at
357 0.944 0.945 0.945 0.945
kemission 605 nm.
358 0.946 0.947 0.948 0.947
359 0.949 0.949 0.949 0.949
360 0.950 0.951 0.950 0.951
361 0.951 0.952 0.952 0.952 in Fig. 6. The flavonoid content in the extract was calculated as
362 0.952 0.952 0.952 0.952 quercetin equivalence. The calculation produced a result of 0.
363 0.952 0.952 0.952 0.952 022 ± 0.002 lg/lL quercetin, which is equivalent to 10 mg per
364 0.950 0.950 0.952 0.951 weight of ethanol extract.
365 0.949 0.950 0.949 0.949
370 0.927 0.928 0.928 0.927 3.6. LCMS/MS quantification
380 0.842 0.842 0.843 0.843
390 0.766 0.766 0.766 0.766
400 0.737 0.738 0.738 0.738 Quantification of A. paniculata plant extracts was analyzed by
LCMS/MS, where the results are presented in Fig. 7A. The
chromatogram showed the presence of glycosylated flavonoids
at the retention peak of 2.77 min and non-glycosylated flavo-
Rf values which suggest the characteristics of certain com- noids found at the retention peak of 3.06 min. Based on the
pounds in certain eluents. The value of Rf in a good TLC is chromatogram results of A. paniculata plant extracts using
typically between 0.2 and 0.8 (Cid-Hernández et al., 2018). LCMS/MS method shown in Fig. 7B, the peaks are seen at
The flavonoid content in A. paniculata plant extracts is shown retention time of 2.77 min, similar to quercetin-3-glycoside
8894 Q. Fardiyah et al.

Fig. 5 HPTLC chromatogram of A. paniculata plant extracts with Rf. 0,61.

Fig. 6 Quercetin standard curve.

(Que-3G) compounds. Therefore, que-3G compounds can be

identified by comparing the retention times (Rt) using standard
compounds.
In Fig. 8, linearity is confirmed by preparing a standard
solution of quercetin-3-glycoside in ethanol at four concentra-
tions. Calibration curves are plotted and determined using
standard data for quercetin-3-glycoside, y = 55874x + 2905.
8 and R2 = 0.9997, which are linear for the specified concen-
tration range. The detection limit was 0.1104 mg/mL and the
quantification limit was 0.3679 mg/mL for que-3G. Quantifica-
tion results indicate that the amount of que-3G present in the
plant extracts of A. paniculata is quite high above the detection
and quantification limits, which further emphasizes the
reliability of this method. The accuracy of measurements is
Fig. 7 Quercetin 3 glycoside standard LCMS/MS chro- within the allowable value. The quercetin-3-glycoside concen-
matogram (Rt: 2.77 min) (A); LCMS/MS chromatogram of A. tration present in the plant extracts of A. paniculata was
paniculata plant extracts: flavonoid glycoside area (Rt: 2.77 min) 9.17 mg/mL.
(B).
New potential and characterization of Andrographis paniculata L. Ness plant extracts as photoprotective agent 8895

Fig. 8 Quercetin-3-glycoside standard curve.

Fig. 9 Sun Protection Factor (SPF) of A. paniculata plant extracts.

3.7. Sun protection factor (SPF) of A. paniculata plant extracts

Table 3 SPF value of A. paniculata plant extracts.

The results of determining the SPF value from A. paniculata Extracts concentration (mL/mL) SPF value
plant extracts are presented in Fig. 9. A. paniculata plant 4 1.52 ± 0.00
extracts have photoprotective abilities evaluated by methods 10 11.80 ± 0.18
developed by Mansur et al. (1986). According to the Food 12 15.42 ± 0.01
and Drug Administration of the United States and European 16 23.68 ± 0.08
Union (FDA), only SPF values that are greater than or equal 20 28.41 ± 0,05
to 15 are suitable for use in pharmaceutical preparations with
photoprotective activity. A. paniculata plant extracts with dif-
ferent concentrations (12, 16 and 20 mL/mL) have satisfying ation absorption at 230 nm and 362 nm. Double-peak phe-
photoprotective activity (15.42, 23.68 and 28.41, respectively), nomenon is also one of the characteristics of Traditional
which are higher than those recommended by the FDA shown Chinese Medicine (TCM) due to enterohepatic circulation
in Table 3. The SPF value of the A. paniculata plant extracts and in vivo isomerization of chemical constituent. Interconver-
being tested depend on the concentration, whereby increasing sion of chemical structure of most of steroidal saponins and
the concentration results in an increase in SPF value. This is triterpenoid leads to the double-peak phenomenon (Mehta
due to the flavonoid content found in the family Acanthaceae and Dhapte, 2016). Phytochemical studies described the pres-
species. Plant extracts that are rich in flavonoids that are effi- ence of flavonoids, and other constituents such as tannins
cient in absorbing ultraviolet radiation usually show two max- and alkaloids. A. paniculata plant extracts describe photopro-
imum peaks of ultraviolet absorption. This is evident in A. tective activity that is significantly correlated with existing fla-
paniculata plant extracts having two peaks of ultraviolet radi- vonoid compounds.
8896 Q. Fardiyah et al.

4. Conclusion Chuah, X.Q., Mun, W., Teo, S.S., 2017. Comparison study of anti-
microbial activity between crude extract of Kappaphycus alvarezii
In summary, Andrographis paniculata L. ness plant extracts were char- and Andrographis paniculata. Asian Pacific J. Tropical Biomed. 7,
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paniculata plant extracts were obtained from phytochemical test, infra- Churiyah, Pongtuluran, O.B., Rofaani, E., Tarwadi, 2015. Antiviral
red analysis, UV characterization, fluorescence identification and and Immunostimulant Activities of Andrographis paniculata.
LCMS/MS characterization. The plant extract of A. paniculata dis- HAYATI J. Biosci. 22, 67–72. https://doi.org/10.4308/hjb.22.2.67.
played excellent photoprotective properties against UV-B and UV-A Cid-Hernández, M., López Dellamary-Toral, F.A., González-Ortiz, L.
radiation due to the presence of flavonoid derivatives present in the J., Sánchez-Peña, M.J., Pacheco-Moisés, F.P., 2018. Two-dimen-
plants, including quercetin. The crude plant extracts of A. paniculata sional thin layer chromatography-bioautography designed to
showed a higher SPF value compared to the value recommended by separate and locate metabolites with antioxidant activity contained
the FDA. The plant extract of A. paniculata has great a potential for on spirulina platensis [www Document]. Int. J. Anal. Chem. https://
use as a photoprotective agent in pharmaceutical preparations. doi.org/10.1155/2018/4605373.
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Declaration of Competing Interest https://doi.org/10.1016/j.soncn.2013.06.008.
Costa, S.C.C., Detoni, C.B., Branco, C.R.C., Botura, M.B., Branco,
The authors declare that they have no known competing A., 2015. In vitro photoprotective effects of Marcetia taxifolia
financial interests or personal relationships that could have ethanolic extract and its potential for sunscreen formulations.
appeared to influence the work reported in this paper. Revista Brasileira de Farmacognosia 25, 413–418. https://doi.org/
10.1016/j.bjp.2015.07.013.
Acknowledgement Diffey, B.L., 2007. A method for broad spectrum classification of
sunscreens. Int. J. Cosmet. Sci. 16, 47–52. https://doi.org/10.1111/
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The author is very grateful to the Directorate General of
Dutra, E.A., Oliveira, D.A.G. da C., Kedor-Hackmann, E.R.M.,
KEMENRISTEKDIKTI (Ministry of Research, Technology Santoro, M.I.R.M., 2004. Determination of sun protection factor
and Higher Education of the Republic of Indonesia) and Insti- (SPF) of sunscreens by ultraviolet spectrophotometry. Revista
tut Teknologi Sepuluh Nopember for providing Domestic Brasileira de Ciências Farmacêuticas 40, 381–385. https://doi.org/
Postgraduate Education Scholarship (BPPDN) with grant 10.1590/S1516-93322004000300014.
number 2903.4/D3/PG/2017 and supporting the research fund- Gabros, S., Nessel, T.A., Zito, P.M., 2020. Sunscreens and Photopro-
ing of this work under project scheme of the Doctoral Disser- tection, in: StatPearls. StatPearls Publishing, Treasure Island (FL).
tation Research (PDD) with grant number 1239/PKS/ Fardiyah, Q., Suprapto, Kurniawan, F., 2018. Fluorescence analysis of
ITS/2020. This research is also partially funded by the Indone- Andrographis paniculata L. ness medicinal plant extract as a
potential protector of ultraviolet radiation. AIP Conf. Proc. 2049
sian Ministry of Research, Technology and Higher Education
(1), 0094–243X. https://doi.org/10.1063/1.5082422.
under WCU Program, managed by Institut Teknologi
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