ARTICLE 1

Papaya (Carica papaya) is one of the most cultivated plants in tropical countries and

the most popular and economically important species among the Caricaceae family. Many in-

vitro and in-vivo studies have demonstrated the medicinal properties of the extracts of papaya

leaves including anti-dengue, anti-plasmodial, anti-cancer, anti-bacteria, hepatoprotection,

anti-inflammatory and antioxidant. To date, few studies are concerned in the investigation of

the composition and biological activities of the constituents of C. papaya leaf. By using high

performance liquid chromatography (HPLC)-based activity profiling, Julianti et al. reported

that flavonoids and alkaloids were the active constituents of C. papaya leaf and suggested the

alkaloid carpaine as the major anti-plasmodial compound. Furthermore, Julianti et al. also

developed and validated a quantitative assay for determination of carpaine in papaya leaves.

In case of flavonoids, several studies have performed the identification of the

flavonoid constituents in C. papaya leaves using ultra performance liquid chromatography-

time of flight-electrospray ioization-mass spectrometry methods. Afzan et al. identified four

flavonoids in the C. papaya leaf extract including quercetin 3-(2G-rhamnosylrutinoside),

kaempferol 3-(2G-rhamnosylrutinoside), quercetin 3-rutinoside, and kaempferol 3-rutinoside.

Nguyen et al. tentatively identified kaempferol β-d-glucopyranoside, luteolin β-d-

glucopyranoside, myricetin 3-rhamnoside, quercetin and rutin. Tan et al. found apigenin,

kaempferol, quercetin, myricetin, isorhamnetin, catechin, hesperitin and naringenin. Using

HPLC, Andarwulan et al. detected the presence of quercetin, kaempferol and apigenin.

Concentration of kaempferol 3-(2G-rhamnosylrutinoside) was much higher than other

flavonoids both in MeOH extract and BuOH fraction.

Plant material
 Leaves of C. papaya were collected from a papaya farm near Pelaihari City, South

Kalimantan Province, Indonesia. Plant species was identified and authenticated at the

Department of Agronomy, Lambung Mangkurat University, and the voucher specimen (No.

C-23) was deposited in the herbarium of Laboratory of Natural Products, Department of

Agro-industrial Technology, Lambung Mangkurat University. The collected leaves of C.

papaya were immediately stored for 6 days in a dark room for airy drying and dried

completely in an oven at 40 °C. Prior to extraction process, the dried plant material was

coarsely powdered using a rotary grinder.

Extraction

Dried leaves of C. papaya (750 g) were extracted three times with MeOH (6 L) under

reflux at 70 °C for 5 h. The extract was filtered and concentrated on a vacuum rotary

evaporator to give a viscous mass (86 g). This MeOH extract (80 g) was suspended in 800

mL H2O and fractioned with 800 mL CHCl3 three times. The residual aqueous layer was

then fractionated with 800 mL BuOH three times, and the BuOH-soluble portion was

concentrated to give the BuOH fraction (25.1 g). The BuOH fraction (24.0 g) was subjected

to silica gel column chromatography (ø 50 mm × 35 cm, SiO2) using CHCl3–MeOH–H2O

(70:30:10, lower phase) as a mobile phase with flow rate of 4.5 mL/min. Totally, 186

fractions (each 50 mL) were collected and monitored by TLC and under UV 254 nm for

combining into eight fraction groups (A–H).

From the BuOH fraction obtained from MeOH extract of C. papaya leaves, seven

flavonoids (quercetin 3-(2G-rhamnosylrutinoside), kaempferol 3-(2G-rhamnosylrutinoside),

quercetin 3-rutinoside, myricetin 3-rhamnoside, kaempferol 3-rutinoside, quercetin and

kaempferol) were successfully isolated through combination of normal and reversed systems

of open column chromatography. Extraction with MeOH was reasonable, because MeOH has
capability to effectively produce greater quantities of flavonoids due to more favorable

partitioning kinetics. BuOH was selected as solvent for fractionation because it has

appropriate polarity to yield a flavonoid-rich fraction. As shown in Table 2, among the

isolated compounds, kaempferol 3-(2G-rhamnosylrutinoside) was obtained in the highest

amount (66.97%).

Table 2. Content of compounds in MeOH extract of C. papaya leaf and its BuOH fraction

(mg/g of lyophilized extract or fractions)

Compounds MeOH extract BuOH fraction

Quercetin 3-(2G- 3.11 25.53

Kaempferol 3-(2G- 7.23 123.18

Quercetin 3-rutinoside (3) 0.97 14.54

Myricetin 3-rhamnoside (4) 0.81 9.78

Kaempferol 3-rutinoside (5) 0.52 10.15

Quercetin (6) < LOQ 0.55

Kaempferol (7) < LOQ 0.19

Total 12.64 183.92

As shown in Table 2, the content of kaempferol 3-(2G-rhamnosylrutinoside) is much

higher than other flavonoids, either in MeOH extract or in BuOH fraction (7.23 and 123.18

mg/g, respectively). Based on the HPLC chromatogram of BuOH fraction, the peak area of

kaempferol 3-(2G-rhamnosylrutinoside) was also dominant compared to other flavonoids. It

papaya leaf.

Flavonoids have been frequently reported as the active substances associated with

antioxidant properties and health benefits. The association of flavonoids with health benefits

is explained by their antioxidant properties that can neutralize free radicals through their

inherent redox properties. Flavonoids work as radical scavengers of lipid peroxidation chain

reactions. They donate an electron to reactive free radical species in the body, neutralizing

their potentially damaging chain reactions in cell chemistry and forming stable phenolic

radical products in the process.

By this experiment, we know that antioxidant flavonoids are abundant in C. papaya

leaf, and kaempferol 3-(2G-rhamnosylrutinoside) was the major one. These results are

important to understand more about the composition of flavonoids in C. papaya leaf and

provide more physical and chemical evidences especially to the previous studies reported the

existence of such flavonoids in C. papaya leaf by liquid chromatography-electrospray

ionization-mass spectrometry-based detection methods.

Seven flavonoids including quercetin 3-(2G-rhamnosylrutinoside), kaempferol 3-(2G-

rhamnosylrutinoside), quercetin 3-rutinoside, myricetin 3-rhamnoside, kaempferol 3-

rutinoside, quercetin and kaempferol were isolated from the leaves of C. papaya. From the

HPLC quantitative analysis, kaempferol 3-(2G-rhamnosylrutinoside) contained the highest

amount among the flavonoids both in MeOH extract (7.23 mg/g) and in BuOH fraction

(123.18 mg/g). The present study reveals that antioxidant flavonoids of C. papaya leaf are

comprised of quercetin, kaempferol, and their certain glycosides, and suggests that

kaempferol 3-(2G-rhamnosylrutinoside) is an important flavonoid of C. papaya leaf because

of its abundance and strong antioxidant activity.

flavonoids in Carica papaya leaf and peroxynitrite-scavenging activity. Asian Pacific Journal

of Tropical Biomedicine, 7(3), 208-213. https://doi.org/10.1016/j.apjtb.2016.12.009

Indonesia has a large variety of plant species. The papaya plant has been used by the

local people as herbal medicine. Papaya belongs to the family Caricaceae. Papaya which is

distributed in Indonesia consists of 2 species, Carica papaya and Carica pubescens. Mountain

papayas (Carica pubescens) spread limitedly and can only adapt in the highland environment.

In Malang area, C. pubescens is found only in the Cangar area (3000 masl). The study was

carried out to determine active compounds, antioxidant activity, and total phenolic of C.

pubescens stem and leaves methanolic extract.

The medicinal properties derived from a plant depend on the content of its

phytochemical constituents and nutritive elements such as minerals. Importantly, the

effectiveness of a plant that is used as a drug depends on the concentration of those nutrient

compounds. Natural products, either as pure compounds or as standardized extracts, provide

many benefits because of the unmatched availability of chemical diversity. Phytochemical

screening is also an essential process to evaluate the medicinal properties of plants. The

bioactive compounds of the plant initially come from leaves, stems, roots, flowers, fruit, or

seeds. For this reason, all parts of plants potentially contain active ingredients.

Several previous studies have shown that the active compounds of aqueous extract of

C. papaya (Caricaceae family) leaves were tannin, saponin, flavonoid, terpenoid, steroid,

alkaloid. However, from those six compounds, the only terpenoid which could not be

detected in extract ethanol papaya leaves. Identification and quantification flavonoid used

chromatographic and spectroscopic methods found 7 flavonoid compounds namely quercetin,

kaempferol, myricetin, quercetin 3-(2G - rhamnosyl-rutinoside), kaempferol 3-(2 G -

rhamnosyl-rutinoside), quercetin 3-rutinoside, myricetin 3- rhamnoside, and kaempferol 3-

rutinoside. The extraction and quantification of carpaine in papaya leaves extract yielded
63% from alkaloid total. The strength of the HPLC (High-Performance Liquid

Chromatography) method that has been done is to identify flavonoids and quantify carpaine

which in the other analyses, e.g., qualitative analysis, it cannot detect the compound

specifically.

Plant material

The plant materials used in this research consisted of the leaves and stems of

mountain papaya (Carica pubescens) which were collected from Cangar, Batu (Indonesia) at

an altitude of 3000 m above sea level. The samples were then deposited in Herbarium unit in

the Biology Department, Universitas Negeri Malang. Before the analysis process, the samples

were washed with water to remove dust at first. Then the leaves and stem of C. pubescens

were dried by the oven at 50 °C for 3 days and crushed into powder form using a dry blender.

Preparation of plant extract

C. pubescens leaves powder was extracted by maceration method by using methanol,

ethyl acetate, and chloroform as a solvent with a ratio 1:10. The mixture was filtered using

Whatman filter paper, and the extracts were evaporated to dryness using a water bath. The

extract obtained was used for phytochemical screening. The C. pubescens leaves, and stem

powder was extracted by maceration method using methanol as a solvent with ratio 1:10 (20

grams the simplicia dissolved in 200 mL methanol). Maceration process was performed at

room temperature for 4×24 hours used shaker at 100 rpm. The mixture was filtered using

Whatman filter paper and was evaporated using a water bath. The obtained leaves and stems

extract were used for antioxidant and total phenol.

The active compounds of papaya leaves extract which has the highest percentage were

alkaloid compounds (carpaine, pseudocarpaine, Dehydrocarpaine I and II). In the same way,
the identified alkaloid compounds in mountain papaya are the same as the active compounds

contained in the Carica papaya leaves. Another active compound is phenolic compounds

(flavonoids) namely kaempferol and quercetin identified in all three types of extracts.

Quercetin and kaempferol are recognized as a source of antioxidants.

Indonesia has a large variety of plant species. The papaya plant has been used by the

local people as herbal medicine. Papaya belongs to the family Caricaceae. Papaya which is

distributed in Indonesia consists of 2 species, Carica papaya and Carica pubescens. Mountain

papayas (Carica pubescens) spread limitedly and can only adapt in the highland environment.

In Malang area, C. pubescens is found only in the Cangar area (3000 masl). The study was

carried out to determine active compounds, antioxidant activity, and total phenolic of C.

pubescens stem and leaves methanolic extract.

The medicinal properties derived from a plant depend on the content of its

phytochemical constituents and nutritive elements such as minerals. Importantly, the

effectiveness of a plant that is used as a drug depends on the concentration of those nutrient

compounds. Natural products, either as pure compounds or as standardized extracts, provide

many benefits because of the unmatched availability of chemical diversity. Phytochemical

screening is also an essential process to evaluate the medicinal properties of plants. The

bioactive compounds of the plant initially come from leaves, stems, roots, flowers, fruit, or

seeds. For this reason, all parts of plants potentially contain active ingredients.

Several previous studies have shown that the active compounds of aqueous extract of

C. papaya (Caricaceae family) leaves were tannin, saponin, flavonoid, terpenoid, steroid,

alkaloid. However, from those six compounds, the only terpenoid which could not be

detected in extract ethanol papaya leaves. Identification and quantification flavonoid used
chromatographic and spectroscopic methods found 7 flavonoid compounds namely quercetin,

kaempferol, myricetin, quercetin 3-(2G - rhamnosyl-rutinoside), kaempferol 3-(2 G -

rhamnosyl-rutinoside), quercetin 3-rutinoside, myricetin 3- rhamnoside, and kaempferol 3-

rutinoside. The extraction and quantification of carpaine in papaya leaves extract yielded

63% from alkaloid total. The strength of the HPLC (High-Performance Liquid

Chromatography) method that has been done is to identify flavonoids and quantify carpaine

which in the other analyses, e.g., qualitative analysis, it cannot detect the compound

specifically.

Plant material

The plant materials used in this research consisted of the leaves and stems of

mountain papaya (Carica pubescens) which were collected from Cangar, Batu (Indonesia) at

an altitude of 3000 m above sea level. The samples were then deposited in Herbarium unit in

the Biology Department, Universitas Negeri Malang. Before the analysis process, the samples

were washed with water to remove dust at first. Then the leaves and stem of C. pubescens

were dried by the oven at 50 °C for 3 days and crushed into powder form using a dry blender.

Preparation of plant extract

C. pubescens leaves powder was extracted by maceration method by using methanol,

ethyl acetate, and chloroform as a solvent with a ratio 1:10. The mixture was filtered using

Whatman filter paper, and the extracts were evaporated to dryness using a water bath. The

extract obtained was used for phytochemical screening. The C. pubescens leaves, and stem

powder was extracted by maceration method using methanol as a solvent with ratio 1:10 (20

grams the simplicia dissolved in 200 mL methanol). Maceration process was performed at

room temperature for 4×24 hours used shaker at 100 rpm. The mixture was filtered using
Whatman filter paper and was evaporated using a water bath. The obtained leaves and stems

extract were used for antioxidant and total phenol.

The active compounds of papaya leaves extract which has the highest percentage were

alkaloid compounds (carpaine, pseudocarpaine, Dehydrocarpaine I and II). In the same way,

the identified alkaloid compounds in mountain papaya are the same as the active compounds

contained in the Carica papaya leaves. Another active compound is phenolic compounds

(flavonoids) namely kaempferol and quercetin identified in all three types of extracts.

Quercetin and kaempferol are recognized as a source of antioxidants.

Rahayu S.E., Sulisetijono1., Umie L. (2019) Phytochemical Screening, Antioxidant Activity,

and Total Phenol Profile of Carica pubescens Leaves from Cangar, Batu-East Java,

Indonesia. IOP Conference Series: Earth and Environmental Science, 276(1), 012022.

https://10.1088/1755-1315/276/1/012022
 ARTICLE 3

Carica papaya. L belongs to the family Caricaceae. Dried leaves extract is shows the

phytoactive biochemical’s Cystatin, Saponins, Carbohydrates, protein, amino acids, steroids,

tocopherol, phenols, flavonoids, cyanogenic glycoside, Quercetin, Kaempferol and

glucosinolates in multivariate analysis. The concentration of 60 ppm contains more

phytoactive molecules are elevated as Cystatin, Quercetin and Kaempferol. Furthermore,

Papaya leaves metabolites Cystatin therapeutically valuable to human nutrition and health

proved the specificity 99.9%.

The present study exposed to analyze the real-time HPTLC fingerprint profiles of

secondary metabolites in methanolic leaf extracts of Carica papaya. It shows the amount of

the secondary metabolites, specifically, cystatin, quercetin, flavonoids, glycosides, saponins

and kaempferol.

Plant materials

The whole plants of Carica papaya.Land the leaf parts were collected from the Field

of Dhanalakshmi Srinivasan Agriculture College, Perambalur. The referral of the plant has

been deposit, Department of Botany, School of Biological Sciences, St. Joseph College

(Autonomous), Trichy was authenticated.

Preparation of extracts

Fresh leaf materials of the whole plant of Carica papaya. Leaves were collected distinctively

in mass, wash down under running tap water to remove stick on dirt trail by rinsing with

deionised water. The papaya leaves were air dried and pulverized in a mechanical grinder

followed by sieving (sieve no.40) to acquire crude powder. Papaya leaf extraction done by

soxhlet method reveals the liquid extracts were concentrated in vacuum or lyophilized to
yield dry extracts. The concentrated extracts were used for HPTLC analysis. 2 μl of the

extracts of Carica papaya. L were employed for HPTLC analysis.

Metabolomic Profiling of Cystatin, Quercetin and Kaempferol validating by HPTLC

Chemical Data Cystatin (mg/g) Quercetin (mg/g) Kaempferol (mg/g)

Recovery (%) 99.76-99.90 97.8-99.82 99.22-99.78

Accuracy – COV

Interday (n=5) 0.25-0.54 0.12-0.48 0.22-0.51

Intraday (n=5) 0.15-0.39 0.11-0.28 0.12-0.36

Specificity (%) 99.87 99.76 99.58

The sample extracts were run along with the standards and it was perceived to

validate the presence of metabolomic substrates and compared with phytochemical

compounds from chromatogram after derivatization overcome in UV 366 nm the peak range,

area and consecutively high in multivariate analysis of papaya leaf supernatant extract

revealed cystatin, quercetin and kaempferol are extensively high in their concentration as

99.9%. During HPLC quantitation, the values of cystatin, quercetin and kaempferol are

remarkably high in specificity. Cystatin shows that metabolomic profile has the

immunosuppressive role which protect antioxidant, gastroinflammation, nephroitic tumor,

antihepatic, antidiabetic, anticancer and neuro defense in cell system. The present study
revealed that phenolic content and flavonoid of C. papaya leaves encompass the quercetin,

kaempferol and their certain glycosides, and imply that kaempferol is an essential flavonoid

of C. papaya leaf because of its large quantity and produce antioxidant activity.

Carica Papaya lead has the defensive role particularly in immune cells and

hypersensitive actions also recover by plants to inflame the systematic diseases such as

tumorigenesis and anti-helminthiasis. The qualitative analysis of methanolic leaf extracts of

C.papaya analyzed through HPTLC confirmed the presence of many secondary metabolites

like Cystatin, Quercetin, Kaempferol, flavonoids, cardiac glycosides, saponins, and Vitamin

C. The well-determined HPTLC profiles also confirm the rate of these metabolites of

medicinal importance which prop up the conventional therapeutic uses of C.papaya leaves.

The profile of HPTLC determining in C.papaya leaves Out of 32 spots, 9 were

identified as flavonoids (3 in leaf extract) in the methanolic extract of papaya leaves.

Flavonoids, the most well-known secondary metabolites from phenolic compounds, mainly

consist of a complex aromatic ring and a heterocyclic ring of glycoside linkage. Alkaloids

and Flavonoids are most active against anti-inflammatory, anticancer, hypersensitivity and

antioxidant properties.35 Cystatin, Quercetin, Kaempferol is a glycosides were prominent in

methanolic extract of papaya leaves which bear out the therapeutic values of this species in

terms of anticancer, high in nutritive value as they would release phenolics on hydrolysis that

are toxic to airborne pathogens.

To conclude, a better perspective of the plant derived metabolites were analyze by

two way methods substantiate that Cystatin, Quercetin and Kaempferol has predominantly

high in chromatogram which bring out the strong antioxidant and used as the ailments for

autoimmune diseases, neurodegenerative and cancer. HPTLC fingerprinting facilitates in

functionally confirming the presence of unlike constituents depending on the polarity of the
building block which are exhibited as a number of resolved bands. Cystatin, quercetin and

kaempferol also exhibit a wide spectrum of cellular activities, including anti-inflammatory

and anticarcinogenic properties.

Alagendran S., Fernández-Saavedra G., Pushpa N., Sahaya Sathish S., Sathish P., Valarmathi

M., Sudha P., Mohanlal V. A., Vijay N. (2020) Metabolomic and Phytonutritive Profiling of

Carica Papaya. L - An In Vitro Study. International Journal of Life science and Pharma

Research. “International Conference on Cancer Research”, 22-27.

http://dx.doi.org/10.22376/ijpbs/ijlpr/SP07/Jan/2020.1-87