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Samajdar & Samajdar                                                            In silico activity of a topical formulation using Piper longum

Online First                                                                                                                            Indian J Pharm Drug Studies | 1

Original Article 

In silico bioactivity prediction and topical formulation of Piper longum root for 
skin cancer

Saptarshi Samajdar, Debojit Samajdar

From, Department of Pharmaceutical Technology, Brainware University, Kolkata, India.

ABSTRACT

Background: Skin cancer is exceedingly common and the incidence is rising rapidly. Although the mortality rate for skin cancer is not as 
large as other cancers but still a steady mortality rate around the world remains. As per various reports, a significant number of skin cancer 
cases have been reported in India mostly from the Northern and Eastern regions. Objectives: Although many treatment strategies for skin 
cancer have been developed over the years, most of them deal with cell toxicity. So, the main objective of this work is to develop a topical 
formulation using an alternate herbal source ie, Piper longum which can provide solution to skin cancer. Methodology: A herbal 
treatment strategy, using Piper longum has been demonstrated by an in silico bioactivity study. Further, a set of topical cream 
formulations using Piper longum was prepared and evaluated. Results: The study yielded three major compounds, Episesamine (-10.4
kcal/mol), Fargesin (-9.6 kcal/mol) & Pellitorine (-9.1 kcal/mol) from Piper longum were highly effective against skin cancer nuclear 
receptor for Vitamin D (1DB1). The toxicity predicted by the ProTox II tool revealed all ligands with 4-5 level of toxicity and very high 
LD50 values can be predicted to be safe for human usage. The evaluation of the cream formulation reveals it to be smooth, semisolid 
nonirritant in nature having high shelf life and spreadability of around 0.3g.cm/s. Conclusions: Hence from the results we can conclude 
that Piper longum is predicted to have many phytochemicals with multitude of anti-skin cancer effects and its formulation was also found 
to be stable, but further, in vitro and in vivo research is required for its usage in humans. 

Key words: Skin Cancer, Molecular docking, Piper longum, nuclear receptor for Vitamin D, ProTox II, Cream formulation.

he skin provides the outer covering of our body covers 
over 16% of the body's mass and protects our inside 
organs from different threats. As the skin is the body's 

most exposed organ, UV radiation, noxious agents, and 
toxicants can all harm it. These hazardous factors cause 
molecular and metabolic stress, resulting in genomic changes in 
skin cells and skin carcinogenesis [1,2]. Human skin cell 
transformation into cancer is a multistep process involving 
initiation, promotion, and advancement that is thought to be 
stimulated by oxidative stress in cells, resulting in 
transformation (into cancer), survival, and metastasis. Exposure 
to ultraviolet radiation (UVR) is a major risk factor for skin 
cancer. Skin cancers are the most commonly diagnosed
malignancies in Western countries, with a significant increase in
the Indian subcontinent due to increased exposure to ultraviolet

Access this article online

Received 14th Apr 2024

Initial Review 31st May 2024

Accepted 05th Jun 2024

Quick Reponses Code

(UV) radiation [3,4]. Skin cancer is defined by an imbalance 
between inadequate apoptosis and excessive cell proliferation 
and survival in the epidermis. Although UV radiation is the 
most common cause of skin cancer, viruses, mutagens in food, 
mutagens in chemicals, and genetic predisposition are also 
factors [5].

To control skin cancer, multiple synthetic and mechanical 
therapeutic strategies have been designed, but many of them reel 
with the problem of cell toxicity [6]. So, natural products from a 
highly regarded Ayurvedic plant Piper longum can act as a 
solution to the problem. P. longum also called Indian long 
pepper or pippali, is a flowering vine in the family Piperaceae, 
cultivated for its fruit, which is usually dried and used as a spice 
and seasoning which makes it safe for human use. Piper longum
is first mentioned in ancient Indian Ayurvedic texts, where its
therapeutic and culinary properties are extensively discussed. It 
___________________________________________________

Correspondence to: Saptarshi Samajdar, Associate Professor, 
Department of Pharmaceutical, Technology, Brainware 
University, 398, Ramkrishnapur Road, Kolkata-700125, India. 
Email: saptarshisamajdar1993@gmail.com

T



Samajdar & Samajdar                                                            In silico activity of a topical formulation using Piper longum

Online First                                                                                                                            Indian J Pharm Drug Studies | 2

arrived in Greece around the sixth or fifth century BCE,
although Hippocrates only mentioned it as a spice and not as a 
medicine [7,8]. In this study, the in-silico evaluation of the skin 
cancer receptor of P. longum is observed as well as preparation 
& evaluation of an herbal topical formulation has been reported.

MATERIALS AND METHODS

Materials: Authenticated roots of the P. longum plant material 
were acquired from Bixa botanicals and other chemicals were 
taken from S.D. Finechem & Spectrochem India. Distilled water 
was used in all the experiments.

Extraction of P. longum: P longum that had been air-dried and 
finely powdered was individually added to the maceration pot 
using petroleum ether, followed by methanol. Under lowered 
pressure and a regulated temperature, the extract was then 
concentrated until it was completely dry before being stored in a 
refrigerator [9].

Ligands preparation and optimization: As reported by 
Sharma et al., the ligands (9) from P. longum root detected using 
GCMS were reported [10]. Three-dimensional structures of the 
ligands were created in Open Babel and saved in SDF format for 
further preparation and molecular docking analysis [11,12].

Preparation and optimization of skin cancer receptor: The 
protein receptor selected for detection of skin cancer potential 
was the crystallographic structures of the skin cancer nuclear 
receptor for Vitamin D (PDB ID: 1DB1), downloaded from the 
protein data bank. The hetatoms as well as selected water
molecules were removed from the protein while adding polar 
hydrogen for charge using BIOVIA Discovery Studio 2021 
visualizer application for its usage in molecular docking [13].

Molecular docking analyses and visualization: The molecular 
docking studies were performed using PyRx application with 
Auto dock Vina tool. The protein was firstly saved in .pdb
format and loaded to turn it into macromolecule. Then the 
suitable ligands were also loaded their energy was minimized 
then using the Auto dock Vina tool the docking study was 
performed. A grid dimension of 61.06 Å x 51.74 Å x 46.44 Å
was chosen for the experiment in order to get the best 
conforming pattern. The intermolecular interactions between the 
ligands obtained from P. longum and 1DB1 protein were 
identified and visualized using the Discovery Studio 2021 Client 
software [14].

Toxicity prediction: The ligands were subjected to toxicity 
prediction using ProTox II software in human cells (https://tox-
new.charite.de/protox_II/). The webserver takes a two-
dimensional chemical structure as input and reports the possible 

toxicity profile of the chemical for 9 models with confidence 
scores [15].

Formulation of Topical Herbal Cream: White beeswax, liquid 
paraffin, and P. longum were all put into the first beaker. Then, 
heat on a water bath to ensure even mixing. An oil phase 
eventually developed after some time. Water, borax, and methyl 
paraben were added to the second beaker. In a water bath, all the 
ingredients were boiled together to produce the aqueous phase. 
The oil phase was continually mixed into the water phase to 
form a semisolid mass. Once the mixture has become 
homogeneous, remove it from the heat and let the cream cool. 
As the liquid-cooled, menthol crystal was added and thoroughly 
mixed. Two formulations, F1 and F2, were created (Table 1)
using various beeswax and medication concentrations [16].

Table 1: Formula of herbal cream
Ingredients F1 F2
P. longum 2.5 g 5g
White beeswax 10 g 7.5g
Liquid Paraffin 22.5 g 22.5g
Borax 0.35 g 0.35g
Water 14.5 g 14.5g
Methyl Paraben 0.15 g 0.15g
Menthol crystal 0.2 g 0.2g

Evaluation of the Cream Formulations: Formulated herbal 
creams were assessed further using the physical parameters 
listed below. Physical characteristics color, odor, consistency, 
and formulation state.Visual inspection allowed for the 
observation of the color for both formulations. The exact 
Pantone shade was confirmed by ColorGrab application [17].
The odor of both F1 and F2 formulations was observed by 
organoleptic evaluations [17]. The state of the cream was 
visually inspected [17]. The consistency of the formulation was 
tested by manually rubbing cream on the hand [17]. A digital pH 
meter (Mettler Toledo 242) was used to measure the herbal 
cream's pH. 100 ml of distilled water was used to produce the 
cream solution, which was then left to sit for two hours. pH was 
determined in three times for the solution and the average value 
was calculated [18]. The spreadability of the cream formulation 
was assessed by sandwiching the sample between two slides and 
compressing it to a constant thickness with a particular weight 
for a particular period. The specified time required to separate 
the two slides was measured as Spreadability. The shorter the 
time taken for separation of two slides results showed better 
spreadability [18]. To calculate spreadability, use the formula below.

Spreadability = ml/t,
Where,
m-Weight of cream on the slide; l- length moved on the slide; t-
time taken



Samajdar & Samajdar                                                            In silico activity of a topical formulation using Piper longum

Online First                                                                                                                            Indian J Pharm Drug Studies | 3

The washability of cream was applied on the skin surface 
and observed under the running water and the observation was 
recorded [19]. The non-irritancy test results for an herbal cream 
formulation were assessed. The preparation lacked irritation and 
redness [19]. The viscosity of the cream was determined with 
the help of Brookfield viscometer LDV230 at 6-100 rpm with 
the spindle no. 63 [19]. The prepared cream was transferred in a
suitable wide-mouth container and its phase separation was. Set
aside for storage there was not any phase separation occurred 
between the oil phase and water phase [19]. Accelerated 
stability testing of prepared formulations was conducted at 40oC
± 1oC for 60 days. The formulations were kept both at room and 
elevated temperature and observed on the 0th, 10th, 30th, and 60th

day for the different parameters [20].

RESULT

In-silico Studies

PyRx docking was utilized to ascertain the binding affinities and 
significant interactions between P. longum-derived
phytochemical ligands and the skin cancer nuclear receptor for 
Vitamin D (1DB1). The binding affinities of the acquired 
ligands and the standard breast cancer treatment Tamoxifen 
were evaluated. Table 2 shows the binding affinity obtained 
from the protein-bound ligands and the standard drug
Fluorouracil [21,22]. The binding affinity of the P. longum
ranged from -9.4 to -4.2 kcal/mol. The molecular interactions 
between the most active ligands and the active site of the breast 
cancer receptor-targeting aromatase inhibitor protein were 
visualized using the Discovery Studio 2021 Client program 
(Figure 1). These samples showed the expected interactions 
with the protein& active-region amino acids, indicating strong 
antagonistic characteristics against the aromatase inhibitor 
protein, which attacks the nuclear receptor for Vitamin D. For 
the protein coded 1DB1, Episesamine had the highest binding 
affinity of -10.4 kcal/mol followed by Fargesin (-9.6 kcal/mol) 
and Pellitorine (-9.4 kcal/mol).

The ligand with values lowest binding affinity was observed 
in Pipericide (-5.5 kcal/mol). As compared to the standard skin 
cancer drug Fluorouracil (-7.8 kcal/mol), the binding affinities 
of three natural compounds (Episesamine, Fargesin, and 
Pellitorine) derived from P. longum were found to be higher 
indicating their future usage in inhibition of skin cancer (Table 
2) [23]. The same set of ligands was studied for their toxicity 
using ProTox II software showed that all the ligands had a 
predicted class 4 to class 5 toxicity with higher LD50 values 
indicating their safe usage in humans (Table 3) [24]. The 
development and evaluation of a multi-herbal cream were the 
focus of the current study. The evaluation criteria for the 

polyherbal pain relief cream, including its viscosity and phase 
separation, spreadability, washability, non-irritancy test, and 
physical examination, were listed under the results in (Table 4).

Table 2: Docking score of P.longum ligands
Ligands Binding Affinity ( G in kcal/mol) 

1DB1
Brachystamide -7.4
Caryophyllene -7.2
Episesamine -10.4
Fargesin -9.6
Pellitorine -9.1
Pipericide -5.5
Piperitine -5.7
Piperine -5.9
Piperlongumine -6.7
Flurouracil -7.8

Figure 1: Interaction diagram of A. Episesamine B. Fargesin

Table 3: Toxicity prediction of ligands
Ligands Level of Toxicity

(1=highly toxic; 6= 
safe)

Predicted LD50
(µg/ml)

Brachystamide 4 760
Caryophyllene 5 5300
Episesamine 4 1500
Fargesin 4 1500
Pellitorine 5 4185
Pipericide 4 760
Piperitine 4 380
Piperine 5 3100
Piperlongumine 4 1160

Evaluation of topical formulation

The color of F1 formulation was observed to be brown orange 
(84831F) and greenish orange (812417F) for F2. The odor of the 
ayurvedically important herbal topical formulations was 
observed to have having common characteristic cream and paste 
smell. Thus this ensures the stability of the formulations with no 



Samajdar & Samajdar                                                            In silico activity of a topical formulation using Piper longum

Online First                                                                                                                            Indian J Pharm Drug Studies | 4

rancid smells. Both the F1 and F2 formulations were shown to 
be in a semisolid state. Their appearance was mostly showing 
plastic flow properties [25-27]. The consistency of the herbal 
topical formulations was seen to be smooth [25]. The pH of the 
F1 cream formulation was found to be 7.2 while the pH of F2 
formulation was found to be 7.06, both of which are neutral with 
F2 having a better pH. No change of pH was observed even after 
four months of testing [28]. It was found that the cream was 
easily spreadable and had moisturizing properties. The exact 
values of spreadability were found to be 0.3±0.06 g.cm/s and 
0.26±0.04 g.cm/s for F1 and F2 respectively. The spreadability 
value remains the same even after 4 months of observation [28].

Table 4: Results of polyherbal cream
Parameter F1 F2

Color
Brown orange 

(84674F)
Pale Brown orange 

(84831F)
Odor Characteristic smell Characteristic smell
State Semisolid Semisolid
Consistency Smooth Smooth
pH 7.20 7.06
Spreadability 0.3±0.06 g.cm/s 0.26±0.04 g.cm/s
Washability Sticky Non-Sticky
Non-irritancy 
test 

Non-irritant Non-irritant

Viscosity 2803±0.32cps. 2689±0.18cps
Phase 
separation 

No phase separation No phase separation

The washability of F1 formulation was shown to be sticky 
but F2 got easily removed from the skin surface thereby 
showing F2 had higher washability, indicating no stain or sticky 
mass on the skin [29]. In irritancy studies, it was discovered that 
none of the formulations cause redness, edema, inflammation, or 
irritation of the skin. There was no sign of redness or other 
ailments even after 4th month of storage. The viscosity of 
formulated cream was determined by Brookfield viscometer at 
20 rpm using spindle no. 63. The viscosity of cream has been 
reported in the range of 2000 to 4999 cps which indicates that 
the cream is easily spreadable by a small amount of shear. The 
formulated creams F1 and F2 show a viscosity of 2803±0.32cps
and 2689±0.18cps respectively [30]. The creams were very 
stable without any kind of phase separation observed. Moreover, 
no phase separation was seen even after four months of storage.
There were no observable changes in any of the parameters for 
the topical cream formulations (F1 and F2) even after 60 days of 
stability testing [19].

DISCUSSION

Skin cancer is very frequent and is becoming more commonplace. 
Even while skin cancer mortality is not as high as that of other 

cancers, skin cancer mortality is nonetheless consistently high 
worldwide. Several sources state that a considerable number of 
skin cancer cases, primarily from the Northern and Eastern parts 
of India, have been documented. So, to combat this, a robust 
treatment strategy is the need of the hour. Although there are 
various synthetic strategies available but most of them faces 
issue of toxicity and multi drug resistance. So, a new strategy 
using P. longum root can find better potential. The 
phytochemicals from macerated extracts were selected for 
molecular docking studies. 

From the set of phytochemicals, three phytochemicals 
Fargesin (-9.6 kcal/mol), Episesamine (-10.4 kcal/mol), and 
Pellitorine (-9.1 kcal/mol) showed maximum skin cancer 
activity even higher than the standard Flurouracil. The toxicity 
prediction data showed the extracts to be safe for human use. 
Thus, from these data it can be a promising anticancer treatment 
strategy [31]. Based on these results, a set of topical 
formulation was designed and the evaluation of topical 
formulation revealed that the color to be of brown color, having 
semisolid state. The pH of the formulation F1 was 7.2 while the 
pH of F2 was 7.06 indicating F2 having a better pH. With F2 
having a lower viscosity and no phase separation and 
nonirritant, non-sticky was selected as a better formulation [32].
Thus with further studies like in vitro or in vivo as well as 
clinical trial, the treatment strategies can be established [33].

CONCLUSION

Hence from the in-silico studies it could be observed that 
multiple compounds of P. longum root like Fargesin, 
Episesamine, and Pellitorine found in P. longum root showed 
better binding affinity as compared to the standard drug in skin 
cancer receptors. All the ligands were found to be safe (Grade 4-
5) in toxicity prediction studies. Overall the compounds showed 
promising results for their usage in skin cancer nuclear receptors 
for Vitamin D. So, using the promising extract topical cream-
based formulations were prepared which were in the range of 
neutral pH and had no irritancy. The viscosity (2689±0.18cps-
2803±0.32cps) and spreadability values had promises for its 
wide use on skin. Moreover, no changes were observed in 60-
day accelerated stability studies, indicating their superior shelf 
life. Further in vitro and in vivo studies are required to provide 
evidence of its usage in humans.

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Samajdar & Samajdar                                                            In silico activity of a topical formulation using Piper longum

Online First                                                                                                                            Indian J Pharm Drug Studies | 5

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How to cite this article: Saptarshi Samajdar, Debojit Samajdar. 
In silico bioactivity prediction and topical formulation of Piper 
longum root for skin cancer. Indian J Pharm Drug Studies. 2024; 
Online First.

Funding: None;                        Conflicts of Interest: None Stated 


