INTRODUCTION:

Bone is tissue providing fundamental foundation to the body along with being a reservoir for calcium and phosphate. It is involved in safeguarding the vital organs, hematopoiesis and serves as lever for muscles. Bone fracture and injuries occurs due to various reasons. This may include traumatic situations like sport injuries, accident; pathological condition such as osteoporosis, osteopenia, osteomalacia and genetic conditions viz osteogenesis imperfecta^1-3. It’s an organ that has an ability to regenerate naturally by proliferation and regeneration of the cell. During the healing process, the progression of the events and cellular interaction is controlled by various stages namely acute inflammatory phase and hematoma formation, soft callus formation, hard callus formation and remodeling. It is treated using bone graft, metal implant, bone remodeling agents, ethnomedicine along with certain analgesics and anti- inflammatory ¹. Globally more than 2.2 million surgeries to correct bone abnormalities in field of orthopedics, neurosurgery and dentistry are performed⁴. Strategies to comprehend the repair mechanism to hasten the osteoinductive properties and decrease the longevity issues including pain, tumors, osteoporosis, rheumatic diseases, etc. need to be developed^5,6. Several studies have the evidence of the plant material being used for facilitating healing process, by enhancing osteogenic property⁷.Enhancement of bone regeneration using phytoconstituents recruited from the plant extract showcased improvised mechanism including several factors like increased calcium, vitamin D3 levels and bone mineral density, decreased bone loss, and reduced oxidative stress and inflammation⁶. Introducing plant material as a promising alternative for bone healing also contributes in reduction in cost, safety and have lesser side effect as compared to synthetic drugs^3.Heterophragma quadriloculare (Roxb.) K.Schum. is a member of the family Bignoniaceae. Generally known as Warras. It is a deciduous tree with compound leaves, mainly found in parts of India including Madhya Pradesh, Gujarat, Maharashtra, Andhra Pradesh, Karnataka and Tamil Nadu^8-12. Traditional utility of Heterophragma roxburgii is found to be as anti-venom, antidiabetic, antifungal, antiseptic, menorrhea, premature ejaculation, and sores. Literature data confirms the leaves of Heterophragma roxburgii are used for medicinal purpose, particularly antidiabetic, skin diseases, analgesic, and antifungal properties. Studies confirms that this plant has metabolites like alkaloids, tannins, phenolics, terpenoids and flavonoids ^13,14. Literature has shown positive effect on bone regeneration by aqueous extract of medicinal plants like Cameroonian plants, Peperomia pellucida using animal model. Drill hole injury model is one of the animal models used for studying the bone regeneration and remodeling⁷.

MATERIALS AND METHODS:

Production of Heterophragma roxburgii extract:

The Heterophragma roxburgii leaves were collected from in and around Mangalore, Karnataka during September 2021. The leaves were authenticated by Dr. Smitha Hegde, Deputy Director, Nitte university Centre for Science education and research, Paneer Campus, Deralakatte, Mangalore, Karnataka. The obtained fresh leaves Heterophragma roxburgii plant were shade dried for 2 weeks in normal room temperature in the college premises until the leaves were free from moisture or water content. The dried leaves were powered (coarse powder) by using an electrical grinder and around 500g of leaves were obtained. The powdered leaves were then immersed in ethanol in a TLC chamber. The stirring of the mixture was done twice daily for seven days. After seven days, the content is filtered using a muslin cloth. The filtrate is then subjected to distillation under reduced pressure to remove the ethanolic fraction. The drug concentrate left behind is further concentrated to obtain thick slurry followed by evaporation to get dry extract. The extract was stored in the desiccator for further usage. Percentage yield was calculated using the formula below:

% Yield = amount of product obtained (in g) /total amount of powder used (in g) X 100

Animals:

Adult healthy Wistar albino rats that weigh about 180-220g and 2-3 months of age was used for the study. The animals were housed in polypropylene cages, maintained under standard laboratory conditions (12 hours light and 12 hours dark cycle; at 25℃ and with 45-55% relative humidity). Standard pellet diet and water ad libitum was provided throughout the course of the study. Animal study was conducted according to CPCSEA, New Delhi, India and the research was granted permission and accepted by IAEC (NGSMIPS/IAEC/AUG-2023/379)

Experimental protocol:

Acute toxicity:

Evaluation of LD50 was performed in order to study the pharmacological effects and drug safety. Acute toxicity study was performed to ascertain the LD50 of ethanolic extract of leaves of Heterophrahma roxburgii. Acute toxicity studies were performed in male albino rats using an up and down method. The test is useful in identifying substances that are probably not very harmful as well as for observing toxicity indicators. Before administering medication, animals must be put on a strict fast (food and drink must be withheld overnight). Each animal's fasting body weight was ascertained, and the dosage was computed based on that weight. After the administration of the extract, animals were monitored for reaction of the extract according to their behavioral changes within every 30 minutes to 4 hours. Survival of animal is monitored after 1 day of administration. As per Irwin’s chart the remarks were compiled Behavior nature: Irritation, anxious, uneasy, visual placing, passivity. Neurological: Motor actions- Touch and pain reactions, gripping power, gait, pinna, and corneal reflex Autonomic observation: Excretion of faeces, urine output, heart beats, respiratory rate, squirming movements^15-17.

Selection of doses:

It was discovered that the ethanolic bark extract of Heterophragma roxburgii, when taken orally a dosage of 2000 mg/kg body weight, was safe. After administration of extract orally, they were monitored for whole day to look for any indications of toxicity. Even after a whole day, the animals were determined to be well tolerated. The extract's safety was confirmed, and there were no signs of toxicity or fatality. Thus, for the current investigation, two dose levels 100 mg/kg and 400 mg/kg body weight were chosen.

Evaluation of bone regeneration and remodeling activity:

The evaluation of the bone regeneration and remodeling activity of Heterophragma roxburgii leaf ethanolic extract will be carried out using X-ray, CT scan, and hematological parameter (Table 1,2). Male Albino wister rats of age 4-7 week weighing 150-200g was used. Animals were divided into 4 groups containing 6 animals each in a group. Animals were given access to water and food ad libitium.Animals were administered the samples through oral route

Table 1. Treatment protocol for the Bone regeneration and remodeling model

Group	Description of group
Group 1 (Standard)	Supplement of Calcium and Vitamin D mixed with food.
Group 2 (Control)	Normal saline- oral
Group 3 (Test Group) - 100mg/kg	Low dose of plant extract- (100mg/kg)-oral
Group 4 (Test Group) -400mg/kg	Higher dose of plant extract- (400mg/kg)- oral

Subsequent day after the induction of the bone defect, the administration of the normal saline and plant extract was done accordingly. Medium and high doses of leaf extract was administrated for 14 consecutive days orally. The animals were the analyzed for the regeneration of the bone defect using X-ray and CT scanning ³.

Artificially induced bone defect:

Under anesthesia such as isoflurane, a 1 cm long straight incision was made in the front skin of mid-femur of rats. Having divided to reveal the femoral bone surface, the periosteum will be stripped, splitting the muscle. The anterior section of diaphysis of one femur was punctured by using a drill machine, resulting in a drillhole bony defect. Immediately after creating the injury, sutures were placed and rats was administered with analgesic and anti-inflammatory drugs like pentazocine to counteract pain and inflammation ³.

Figure 1: Artifically indcued bone defect

Estimation of bone regeneration and remodeling activity.

Hematological parameters

Before the administration of the doses and after the treatment, blood was withdrawn by prick in tail. Automated blood cell count will be conducted (machine model MEK6550K). Calcium, phosphorous, alkaline phosphate and WBC were evaluated.

Radiography of the bone:

X-ray

X-ray was taken after inducing the bone defect and repeated after the completion of the treatment course to check for the bone regeneration and remodeling at an interval of 7 and 14 days. The animal was anesthetized and placed on the X- ray table in antero posterior view. It was to radiations for 5-10 milliseconds. The factors of X-ray generator are the maximum voltage 125kV, focal spot 2.0/1.0 and permanent filtration 0.9 Al/75 was previously set. The exposure parameters were 45 kVp, 8mAs has been set and the collimation was done [unit model number E7239X]. X-ray images were procured.

CT scan:

CT scan was done to evaluate bone regeneration and remodelling.

CT scan protocol: The animals were anaesthetized using suitable anaesthetizing agents. Rats were placed in antero posterior view on the examination table. According to the height and weight of the respective animal under examination, the CT parameters [ Kv-140, mA- 150, kVp- 120, exposure time 800 milliseconds] were set. Scanning was done and images were procured for further estimation.

Statistical analysis:

Data were expressed in the form of mean ±SEM and analyzed by ANOVA followed by Dunnett’s test using GraphPad Prism software. p value <0.05 was considered as statistically significant.

RESULTS:

Maceration: Leaf extract of Heterophragma roxburgii was prepared as per the method mentioned in the methodology section. A total of 269 g of dry, coarsely powdered leaf was macerated with ethanol (95%). The percentage yield is found to be 9.29% i.e., Percentage yield = 25 x100 /269

Therefore, the percentage yield using the maceration method = 9.29%

Acute toxicity study:

It was discovered that the ethanolic bark extract of Heterophragma roxburgii, when taken orally at a dosage of 2000 mg/kg body weight was safe. After oral dose of the extract, they were monitored for a whole day to look for any indications of toxicity. Even after a whole day, the animals were determined to be well tolerated. The extract's safety was confirmed, and there were no signs of toxicity or fatality. Thus, for the current investigation, two dose levels 100 mg/kg and 400 mg/kg body weight were chosen.

Effects of Heterophragma roxburgii extract on hematological parameters:

A summary of Heterophragma roxburgii's effects on rats' differential blood counts is provided. Plant extract at 100mg/kg subsequently lead to the increase in number of White blood count [WBC] as compared to control group. Extract dose of 400mg/kg showcased a significant rise in WBC count. At dose of 400mg/kg of the fractured rat there was significant increase in the PLT and MPV values when compared to the control group. Similarly, the values of MCH, MCHC, MCV were seen to be significantly increased at the dose of 400mg/kg when compared to control group.

Table 2: Summary of the differential blood count

Groups	Control	Standard	100mg/kg	400mg/kg
WBCs (103/uL)	11.28 ± 0.09b	12.21 ± 0.05a	16.07 ± 0.06a,b	16.72 ± 0.9a,b
RBCs (103/uL)	3.61± 0.08b	5.11 ± 0.06a	4.18 ± 0.06a,b	4.21 ± 0.07a, b
HGB (g/dl)	13.53 ± 0.12	13.52 ± 0.11	13.20 ± 0.07	15.37 ± 0.14a,b
HCT (%)	42.15 ± 0.08	42.37 ± 0.08	42.80 ± 0.05a,b	45.73 ± 0.13a,b
MCV (fl)	52.28 ± 0.09	52.07 ± 0.05	52.72 ± 0.29	59.97 ± 0.12a,b
MCH (Pg)	18.08 ± 0.07	17.75 ± 0.09	18.53 ± 0.14b	20.77 ± 0.12a,b
MCHC (g/dl)	34.2 ± 0.07b	35.22 ± 0.08a	37.52 ± 0.11a,b	40.08 ± 0.07a,b
PLT (103 /uL)	472.7 ± 1.35b	469.2 ± 0.79a	495.8 ± 1.60a,b	515 ± 1.36a,b
MPV (fl)	6.2 ± 0.06	6.55 ± 0.09	11.8± 0.16a,b	14.45 ± 0.09a,b

“Values were expressed as Mean±SEM ,(n=6). a = p˂0.05 is statistically significant compared to control; b=p˂0.05 is statistically significant compared to standard. WBC = White blood cells, RBC = Red blood leaves, HGB =hemoglobin, HCT= Hematocrit, MCV= Mean corpuscular volume, MCH= Mean corpuscular hemoglobin, MCHC= Mean cell hemoglobin concentration, PLT= Platelets, MPV= Mean platelet volume”

Effect of Heterophragma roxburgii extract on serum calcium concentration:

The fractured rat which was given a standard drug exhibited drastic rise in the serum calcium levels post two weeks of the procedure. The animals which were given different doses of plant extract i.e. 100mg/kg and 400mg/kg had slightly lower levels of serum calcium levels as compared to standard group. However, the test group which was given plant extract of 400mg/kg led to significant increase in serum calcium level as compared to the fractured rats of control group. It is to be noted that serum calcium levels were not significantly modified at a dose of 100mg/kg as compared to the control group.

Figure 2: Estimation of serum calcium levels.

Data obtained was analyzed by one way ANOVA followed by Dunnet test using GraphPad computer software version 9. Values were expressed as Mean±SEM,(n=6). a = p˂0.05 is statistically significant compared to control; b=p˂0.05 is statistically significant compared to standard

Estimation of the aqueous extract of the Heterophragma roxburgii on serum phosphorous concentration:

Effects of the aqueous extract of Heterophragma roxburgii on serum phosphorus concentrations is summarized in the figure. Fractured standard rats showed significant fall in serum phosphorous levels . The rats receiving the plant extract of the doses of 100mg/kg and 400mg/kg showcased a decrease in the serum phosphorous levels. The test groups showed significant fall in the serum phosphorous levels. It was noted that plant extract dose of 100mg/kg had less to no difference as compared to standard group. But the test group of fractured rats administered with plant extract dose of 400mg/kg showed a significant decrease in the serum phosphorus levels as compared to the control group and standard group

Figure 3: Estimation of serum phosphorous levels.

Effect of Heterophragma roxburgii extract on serum Alkaline phosphatase [ALP] concentration:

The fractured bone rat administered with standard drug showed a significant increase the levels of serum AP concentration as compared to the control group. The animals who received the plant extract of 100mg/kg and 400mg/kg showed a significant increase in the levels of serum AP concentration as compared to that of control group. Group administered with 100mg/kg of the plant extract did not show significant levels of serum AP concentration change when compared to the standard group (Fig 4). Animals with bone injury showed an increase in the serum AP concentration in comparison to the control group.

Figure 4: Estimation of Alkaline phosphatase levels.

Data obtained was analyzed by one way ANOVA followed by Dunnet test using GraphPad computer software version 9. Values were expressed as Mean±SEM (n=6). a = p˂0.05 is statistically significant compared to control; b=p˂0.05 is statistically significant compared to standard”

Effects of Heterophragma roxburgii on bone callus formation in rats: Radiography

X-ray

Bone X-ray data demonstrated that Heterophragma roxburgii at doses of 100, and 400 mg/kg caused a dose-related reaction in fractured rats. It was noted that drill hole diameter decreased progressively. The rats that were given dose of 400 mg/kg extract showed complete closure of the hole whereas the rats administered with the dose of 100mg/kg almost healed. The progression of healing was seen better in rats administered plant extract of dose 400mg/kg and 100mg/kg as compared to control group. The photographic image of the rats fractured bone demonstrated a dose dependent bone healing through deposition of callus formation. The callus formation was discovered to be denser in animal administered with 400mg/kg of the dose than the rat administered with plant extract at dose of 100mg/kg. It was noted that at 100mg/kg callus formation was slightly less dense.

Figure 5: X-ray images of the various group

CT SCAN:

CT scan was done post the administration of the plant extract after 14th day. It images obtained showed that the animals given the plant extract of 400mg/kg and 100mg/kg have demonstrated healing according to the doses respectively. Whereas the control group showed minimal amount of healing. Fractured bone treated with 400mg/kg of the plant extract was fully healed whereas the one treated with 100mg/kg showed reduction in diameter which was observable. It draws attention that there is dose dependent reduction in the diameter of the drill hole when compared to control group.

Figure 6: CT scan images of the various groups

DISCUSSION:

Heterophragma roxburgii is used for the wound healing but its efficacy for the bone regeneration has not been evaluated ^18-21. There is no current pharmacological active agent available to treat the fractures. There is a need to develop strategies to comprehend the repair mechanism to hasten the osteoinductive properties and decrease the longevity issues including pain, tumors, osteoporosis, rheumatic diseases, etc. Therefore, the focus towards introducing plant material as a promising alternative for bone healing also contributes in reduction in cost, safety and have lesser side effect as compared to synthetic drugs³.

In this study we conducted the phytochemical analysis of the plant extract.it was noted that it has the presence of flavonoids, steroids, phenols, triterpenoids. These were in accordance to Bhavik kumar H Satani, et al ^13,14. These phytoconstituents further help in the stem cells growth and development ²². This suggests us that administration of the plant extract of Heterophragma roxburgii to the fractured bone rats will help in the bone regeneration. Flavonoid is an osteogenic substance which is able to encourage osteoblast recruitment at the site of damage and boost osteoblast activity and quantity ²³. Osteoid or soft callus, which fills the fracture gap, is quickly deposited as a result of accelerated osteoblast activity. This callus later mineralizes and gives the bone weight.

Hematological analysis was carried out in all the study groups. The results revealed that there was increase in the WBC count in the group administered with 100mg/kg and 400mg/kg. This suggested that there was in inflammatory process which has been initiated. Thrombocytes are known to stimulate bone regeneration, and mean platelet volume (MPV) is a useful indicator of their activation and activity. It was noted that there is significant increase in the MPV levels in the rats administered with 100mg/kg and 400mg/kg of the plant extract. This finding implies that the plant extract can hasten the migration and proliferation of osteogenic cells in injured rats, hence promoting bone regeneration. The rise in MPV also attribute the fact that the plant extract can affect thrombocyte activation and function, which may be advantageous in the event of a fracture. Calcium is important in the bone healing process and the reports suggest that there is dose dependent increase in the serum calcium which may be due to the plant extract. There was a significant increase in the levels of calcium in the rats administered with 100mg/kg and 400mg/kg of the plant extract. This may indicate that it may have helped in the mineralization of the bone. Phosphorous is another factor that will help analyze the bone regeneration. The animals administered with 100mg/kg and 400mg/kg of the plant extract showcased fall in the serum phosphorous levels. This maybe the indication of increased in the mineralization which is indicative of decreased phosphorous levels calling it has hungry bone ^24,25. Assessing the clinical development of a healing process can also be facilitated by the identification of particular serum biological marker of bone growth, such as alkaline phosphatase. During the maturation stage of bone production, osteoblasts release alkaline phosphatase which mediates bone synthesis and fracture repair ^{26 .}Plant extract administered to the treatment group of 100mg/kg and 400mg/kg showed a significant increase in the level of alkaline phosphatase in the serum indicating increase in the osteoblast activity.

Further radiological evaluation was done to confirm the healing process of the fractured bone. X-ray examination of the fractured bone showcased that the bone was completely healed with the dose of 400mg/kg of the plant extract to the treatment group. It was seen that callus was formed completely filling the fracture. Additionally, CT scan was done for further confirmation of the healing process of the bone. It showcased the complete closure of the fractured bone treated with 400mg/kg of the plant extract administered to the treatment group. It was noted that the animal administered with 100mg/kg of the plant extract almost healed showing reduction in diameter which was observable. This suggests that there is dose dependent healing that has taken place. It also indicates that 400mg/kg of the plant extract has more powerful healing effect as compared to the lower dose.

Both X ray and CT scan confirmed the healing of bone administered with 100mg/kg and 400mg/kg of the plant extract which was similarly seen in Ngueguim Tsofack Florence.

CONCLUSION:

The current study was directed to evaluate the bone regeneration and remodeling activity of Heterophragma roxburgii leaves ethanolic extract. This was carried out by developing a fracture using a drill hole method. The preliminary phytochemical tests revealed the presence of various types of phytochemicals such as phenols, steroids, triterpenoids and flavonoids. The experimental design included four groups namely control, standard, treatment group which are 100mg/kg and 400mg/kg. The control group was administered with normal saline, standard group was administered with Calcium and Vitamin D supplements. The bone regeneration ad remodeling activity was evaluated using radiology estimation like X-ray and CT scan. Further hematological analysis was carried out which included various parameters like calcium, phosphorous, alkaline phosphatase, etc which hasten the bone regeneration process. The plant extract caused increase in the WBC, MPV, calcium, alkaline phosphatase levels and decrease in phosphorous levels showcasing healing process. It was seen there was dose dependent healing of the fractured bone administered with 100mg/kg and 400mg/kg of the plant extract. Regeneration of the bone was better in animal administered with 400mg/kg of the plant extract as compared to 100mg/kg. whereas, it was observed that animal administered with 100mg/kg of the plant extract showcased more better healing as compared to the control group. From the study results it can be concluded that the ethanolic extract of Heterophragma roxburgii leaves has significant bone regeneration and remodeling activity in a dose dependent manner.

ACKNOWLEDGEMENT:

The Investigators is thankful to NGSM Institute of Pharmaceutical Sciences for providing all the facilities to perform the present study effectively.

CONFLICT OF INTEREST:

Author has no conflict of interest to publish this research paper.

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Received on 07.04.2025 Revised on 10.10.2025

Accepted on 14.02.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3257-3264.

DOI: 10.52711/0974-360X.2026.00464

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